Apparatus and method for separating cells or cell fragments

Abstract

【Solution】 The present disclosure provides a novel apparatus and method for separating cells or cell fragments by finely adjusting the flow rate in a microchannel by utilizing the combined force of capillary force and gravity without using an external pump. The apparatus and method of the present disclosure are unexpectedly effective in capturing various target cells, including rare biological particles such as B cells, using only a small amount of blood sample.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 367,966, filed on July 8, 2022. The entire content of that application is hereby incorporated by reference herein.

[0002] The present disclosure relates to devices and methods for separating cells or cell fragments.

Background Art

[0003] There are numerous clinical implementation plans that can perform multiple events such as cell concentration, imaging, cell counting, and biomarker measurement using innovative all - in - one lab - on - a - chip (LOAC) systems. Microchannels are one of the new fields for achieving such goals. Microchannels control hydrodynamics using small channels. These microchannels are used in various applications such as portable point - of - care (POC) medical diagnostic systems, separation, purification, sorting, chemical / biological reactions, and detection of target cells or molecules. The fluid behavior in microchannels is different from that in large - channel systems. In large channels, turbulent flow is dominant, so the flow is random. In contrast, in microchannels, laminar flow is formed due to their small size, and such laminar flow has advantages in controlling fluids compared to large channels in the above applications.

[0004] Despite the above advantages, there are still several challenges in microchannels, such as the difficulty in maintaining fine adjustment of the flow rate within the microchannels, which is important for achieving the experimental goals. The flow rate within the microchannels can be controlled by different methods or factors. For example, the flow rate of the microchannels is controlled by a small mechanical pump. When using a mechanical pump, the microchannel system becomes large, and complex piping with various valves is required for the entire system. Furthermore, the mechanical pump for controlling the flow rate is expensive because manufacturing small mechanical devices is difficult engineering-wise. The flow rate is also controlled by the surface characteristics of the materials used in the manufacture of the microchannels, as well as the dimensions and structure of the microchannels. In materials with high hydrophobicity, the cohesive force between fluid molecules becomes stronger than the adhesive force of the fluid to the surface of the microchannels, resulting in a decrease in the flow rate. The smaller the channel, the stronger the capillary action, and thus the flow rate increases. The chemical and physical properties of the injected fluid also play an important role. For example, in a highly viscous liquid sample, the flow rate decreases.

[0005] Another problem with microchannels is their low efficiency in separating cells, especially those present in low abundance. For example, since B cells in blood are rare, it has been difficult to separate B cells from a mixture of red blood cells (RBCs) and other cells. B cells are part of the immune system, produced from pluripotent stem cells in the bone marrow and remaining in the bone marrow until they mature. B cells are responsible for antibody production. B cells bind to specific antigens and become activated plasma cells, secreting large amounts of antibodies. B cells are contained in about 2% - 10% (about 400 - about 7,000 cells) in 10 μL of human adult blood, which is only 3% - 15% in human peripheral blood mononuclear cells (PBMCs). In contrast, there are about 50 million RBCs in 10 μL of human adult blood. Many techniques have been applied to separate RBCs from various populations of white blood cells (WBCs). Existing sorting processes include magnetic separation using columns, density gradient centrifugation, and cell separation using bubbles. These methods have improved the quality of WBC preparations, but these methods require large blood samples (i.e., 500 μL - 5 ml) and about 25 ml of wash buffer to remove unwanted biological particles, including RBCs, and do not meet the requirements for home use point-of-care (POC) diagnostic applications. For many WBC separation applications, an additional concentration protocol needs to be utilized before specific cell separation. However, methods such as Ficoll gradient separation and RBC lysis protocols require additional time and large amounts of buffer.

[0006] Therefore, there is an urgent need for an improved cell separation system that can effectively capture target cells or cell fragments using only a small amount of sample. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] The present disclosure addresses the above-described needs in various aspects. In one aspect, the present disclosure provides a microchannel cartridge for separating cells or cell fragments. In some embodiments, the microchannel cartridge includes a first layer, a second layer, and a microfluidic channel layer having microchannels configured to separate cells or cell fragments in a fluid sample, and the microfluidic channel layer is disposed between the first layer and the second layer.

[0008] In some embodiments, the microchannels include an inlet, an outlet, a first flow path in fluid communication with the inlet, a cell collection chamber provided downstream of the first flow path and in fluid communication with the first flow path, and a second flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the outlet.

[0009] In some embodiments, when the inlet of the microchannel cartridge is disposed below the outlet, the microchannel cartridge is configured to load the fluid sample into the inlet by gravity by delaying the passage of the fluid sample into the second flow path, and when the inlet is disposed above the outlet, the microchannel cartridge is configured to separate cells or cell fragments in the fluid sample by gravity by facilitating the passage of unwanted cells or cell fragments into the second flow path.

[0010] In some embodiments, the microchannel cartridge further includes a waste chamber provided downstream of the cell collection chamber and in fluid communication with the second flow path.

[0011] In some embodiments, the microchannel cartridge further includes a third flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the waste chamber. In some embodiments, the width of the third flow path is greater than the width of the first flow path or the second flow path.

[0012] In some embodiments, the microchannel cartridge further has a magnetic member disposed in or near the inlet or the cell collection chamber, and the magnetic member is configured to apply a magnetic force to the fluid sample received from the inlet. In some embodiments, the magnetic member comprises magnetic beads, magnetic particles, magnetic strips, or a combination thereof. In some embodiments, the magnetic member has a cube magnet, and the vertices of the cube magnet are disposed below and facing the cell collection chamber. In some embodiments, the vertices of the cube magnet are disposed below the cell collection chamber.

[0013] In some embodiments, the microchannel cartridge further has a pretreatment solution that occupies at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 100%) of the microchannels. In some embodiments, the pretreatment solution comprises distilled water, deionized water, or a phosphate buffered saline (PBS) buffer. In some embodiments, the pretreatment solution comprises a phosphate buffered saline (PBS) buffer.

[0014] In some embodiments, the microfluidic channel layer has a polymer thin film. In some embodiments, the polymer thin film includes ethylene vinyl acetate (EVA) polymer, acrylic, acrylonitrile butadiene styrene (ABS) polymer, aromatic thermoplastic polyester (e.g., polyacrylic acid), polycarbonate (PC), polydimethylsiloxane (PDMS), polyglycolic acid (PGA), polylactic acid (PLA), polystyrene (PS), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), thermoplastic elastomer (TPE), thermoplastic polyurethane (TPU), natural or synthetic rubber, and mixtures of two or more thereof. In some embodiments, the polymer thin film includes styrene-butadiene-styrene (SBS), styrene-isoprene-styrene (SIS), polypropylene coated with a pressure-sensitive adhesive, or combinations thereof.

[0015] In some embodiments, the microfluidic channel layer is bonded between the first layer and the second layer using thermal lamination or an adhesive.

[0016] In some embodiments, the first layer or the second layer is configured to enable real-time detection of a sample within the cell collection chamber. In some embodiments, the first layer or the second layer is a glass layer. In some embodiments, the first layer or the second layer has a microscope slide glass.

[0017] In some embodiments, the second flow path has a serpentine flow path or a straight flow path. In some embodiments, the microchannel cartridge further has a coating attached to at least a portion of the first flow path and / or the second flow path. In some embodiments, the coating includes chitosan (e.g., neutral chitosan, chitosan salts, chitosan derivatives), chitin, polymethyl methacrylate (PMMA), silicone, polystyrene (PS), polysaccharides (e.g., nonionic, ionic, cross-linked polysaccharides), poly-D-lysine, streptavidin, collagen, polyurethane, epoxy, or combinations thereof.

[0018] In some embodiments, the coating includes one or more antibodies. In some embodiments, the one or more antibodies are associated with quantum dots. In some embodiments, the one or more antibodies are biotinylated. In some embodiments, the one or more antibodies include an anti-C4d antibody or an antibody that specifically binds to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268.

[0019] In some embodiments, the first flow path or the second flow path has a height of about 50 μm to about 500 μm. In some embodiments, the first flow path or the second flow path has a width of about 2 mm to about 20 mm. In some embodiments, the microchannel has a length of about 25 mm to about 75 mm.

[0020] In some embodiments, the cell collection chamber has a rectangular, oval, or diamond shape. In some embodiments, the cell collection chamber has a diamond shape. In some embodiments, the cell collection chamber has an area of about 9 mm 2 to about 225 mm 2 .

[0021] In some embodiments, the inlet or the outlet has an absorbent material disposed therein. In some embodiments, the absorbent material has a pore size in the range of about 100 μm to about 500 μm. In some embodiments, the absorbent material comprises absorbent fibers or sponges. In some embodiments, the absorbent material comprises cotton, polyester, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), or combinations thereof.

[0022] In some embodiments, the absorbent material is configured to generate a capillary force greater than that of the second flow path to prevent air bubbles from entering the inlet.

[0023] In some embodiments, the microchannel cartridge has a wet filter paper in fluid communication with the outlet.

[0024] In some embodiments, the fluid sample comprises whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, plasma, or combinations thereof. In some embodiments, the fluid sample comprises blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets.

[0025] In another aspect, the present disclosure also provides a kit having one or more microchannel cartridges described herein, and optionally a buffer or instructional materials. In some embodiments, the kit further comprises an immunoassay reagent. In some embodiments, the immunoassay reagent comprises an antibody.

[0026] In yet another aspect, the present disclosure further provides a microchannel cell separation device. The microchannel cell separation device includes one or more microchannel cartridges described herein and a rotating member configured to change the angle of the microchannel cartridge, whereby the flow rate of the fluid sample in the microchannel is adjusted.

[0027] In some embodiments, the rotating member has a holding device for the microchannel cartridge. In some embodiments, the microchannel cartridge is removably attached to the holding device. In some embodiments, the rotating member has an angle indicator with one or more marks indicating a loading position, a standby position, and / or a sorting position. In some embodiments, the rotating member is configured to rotate the microchannel cartridge continuously or intermittently to control the flow rate of the fluid sample. In some embodiments, the rotating member is driven by a motor.

[0028] In some embodiments, the microchannel cell separation device further includes one or more compression springs and / or one or more washers disposed between the base and the rotating member.

[0029] In another aspect, the present disclosure further provides a method for separating cells or cell fragments in a fluid sample. In some embodiments, the method comprises: (a) disposing the microchannel cartridge described herein at a sample loading angle; (b) introducing the fluid sample into the microchannel through an inlet of the microchannel; (c) performing a first gravitational separation by incubating the fluid sample for a first period; and (d) disposing the microchannel cartridge at a sorting angle for gravitational separation for a second period to perform a second gravitational separation, wherein the second gravitational separation induces a flow of target cells or cell fragments into the cell collection chamber.

[0030] In some embodiments, the method further comprises loading a pretreatment solution into the microchannel prior to the step of introducing the fluid sample. In some embodiments, the pretreatment solution is selected from distilled water, deionized water, and phosphate buffered saline buffer.

[0031] In some embodiments, the method further comprises loading a wash solution into the microchannel to wash the cells or cell fragments after the step of introducing the fluid sample. In some embodiments, the wash solution comprises phosphate buffered saline buffer.

[0032] In some embodiments, the method comprises detecting cells or cell fragments in the cell collection chamber using Raman spectroscopy, surface enhanced Raman spectroscopy (SERS), fluorescence microscopy, and magnetic resonance (MR). In some embodiments, the detecting step comprises counting the number of cells.

[0033] In some embodiments, after the step of washing the cell or the cell fragment, the method further comprises the step of disposing a wet absorbent material at the outlet, the wet absorbent material being in fluid communication with the outlet and facilitating the removal of unwanted cells or cell fragments. In some embodiments, the wet absorbent material is a wet filter paper. In some embodiments, the method further comprises the step of applying hot air or room temperature air to the wet absorbent material to gradually evaporate the liquid from the wet absorbent material in order to facilitate the removal of unwanted cells or cell fragments.

[0034] In some embodiments, the method further comprises the step of adding an immunological reagent to the fluid sample prior to the step of introducing the fluid sample. In some embodiments, the immunological reagent comprises one or more antibodies. In some embodiments, the one or more antibodies are associated with quantum dots. In some embodiments, the one or more antibodies are biotinylated. In some embodiments, the one or more antibodies comprise an anti-C4d antibody or an antibody that specifically binds to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268.

[0035] In some embodiments, the fluid sample comprises whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, or plasma. In some embodiments, the fluid sample comprises blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets.

[0036] In some embodiments, the fluid sample comprises magnetically labeled cells.

[0037] In some embodiments, the sample loading angle is an angle of about -90 degrees to about 0 degrees below the horizontal line. In some embodiments, the sample loading angle is an angle of about 90 degrees to about 0 degrees above the horizontal line.

[0038] The above summary is not intended to define all aspects of the present disclosure, and additional aspects are described in other sections such as the following detailed description. The entire specification is intended to be associated as a unified disclosure, and it should be understood that combinations of features described herein are contemplated in their entirety, even if not described together in the same sentence, paragraph, or section of the specification. Other features and advantages of the present invention are believed to become apparent from the following detailed description. However, while the detailed description and specific examples illustrate certain embodiments of the present disclosure, it should be understood that they are for illustrative purposes only, as various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0040] The present disclosure provides a novel apparatus and method for separating cells or cell fragments by finely adjusting the flow rate in a microchannel by utilizing the combined force of capillary force and gravity without using an external pump. The disclosed apparatus and method are unexpectedly effective in capturing various target cells, including rare biological particles such as B cells, using only a small amount of blood sample (e.g., 20 μL or less). The disclosed apparatus and method demonstrated up to approximately 97-fold enrichment of B cells with only 10 μL of whole blood compared to a magnetic sorting system using a conventional pump. The disclosed apparatus and method can be implemented in a portable all-in-one cell separation system that enables streamlined cell separation, cell counting, and imaging. The apparatus and method disclosed herein have various applications, including (but not limited to) immunology, cancer, neuroscience, stem cells, and nanotechnology.

[0041] Apparatus and Detector for Separating Cells or Cell Fragments Thus, in one aspect, the present disclosure provides a microchannel cartridge for capturing target biological particles such as cells or cell fragments. In some embodiments, the microchannel cartridge can include a first layer, a second layer, and a microfluidic channel layer including microchannels configured to separate cells or cell fragments in a fluid sample (an intermediate layer component between the first layer and the second layer), and the microfluidic channel layer is disposed between the first layer and the second layer.

[0042] In some embodiments, the microchannel can include an inlet, an outlet, a first flow path in fluid communication with the inlet, a cell collection chamber provided downstream of the first flow path and in fluid communication with the first flow path, and a second flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the outlet.

[0043] In some embodiments, when the inlet of the microchannel cartridge is disposed below the outlet, the microchannel cartridge is configured to delay the passage of the fluid sample into the second flow path, thereby enabling the fluid sample to be loaded into the inlet by gravity. When the inlet is disposed above the outlet, the microchannel cartridge is configured to facilitate the passage of unwanted cells or cell debris into the second flow path, thereby enabling the cells or cell debris in the fluid sample to be separated by gravity.

[0044] The term "cell" or "cell population" refers to a sample containing more than one cell or more than one type of cell. For example, a blood sample from a subject is a population of white blood cells and red blood cells. A cell population can also include a sample containing a plurality of substantially homogeneous cells, such as those obtained by a cell culture method of a continuously subcultured cell line. The term "cell" as used in the context of a biological sample generally encompasses samples of a size comparable to that of individual cells and includes (but is not limited to) vesicles (such as liposomes), cells, virus particles, and substances bound to small particles such as beads, nanoparticles, and microspheres. In some embodiments, the cells are blood cells, cord blood cells, bone marrow cells, red blood cells, white blood cells, lymphocytes, epithelial cells, stem cells, cancer cells, tumor cells, circulating tumor cells, progenitor cells, cell precursors, cord blood stem cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, cord-derived cells, adipose tissue-derived cells, matrix cells in stromal vascular fraction (SVF), cells in amniotic fluid, cells in menstrual blood, cells in cerebrospinal fluid, cells in urine, bone marrow stem cells, peripheral blood stem cells, CD34+ cells, colony-forming cells, T cells, B cells, nerve cells, immune cells, dendritic cells, megakaryocytes, immobilized bone marrow cells, platelets, sperm, eggs, oocytes, pathogenic bacteria, microorganisms, bacteria, fungi, yeast, protozoa, viruses, organelles, nuclei, nucleic acids, mitochondria, micelles, lipids, proteins, protein complexes, cell debris, parasites, lipid droplets, multicellular organisms, spores, algae, clusters, aggregates of the above, industrial powders, polymers, powders, droplets of emulsions, dust, spherical particles (e.g., microspheres), microparticles, and colloidal dispersions (e.g., colloids).

[0045] As used herein, "cell fragment" refers to a part of a cell, such as an organelle or a part thereof, including (but not limited to) the nucleus, endoplasmic reticulum, mitochondria, Golgi apparatus, etc. The cell fragment can include vesicles such as inner or outer vesicles, or mixtures thereof. Preparations containing cell fragments can be prepared using methods known in the art. "Population of cell fragments" refers to a sample containing more than one cell fragment or more than one type of cell fragment. For example, a population of cell fragments can include mitochondria, nuclei, microsomes, and a part of the Golgi apparatus formed during cell lysis.

[0046] As used herein, "microfluidics" refers to an apparatus having one or more fluid channels, chambers, or conduits with at least one internal cross-sectional dimension, such as depth, width, length, diameter, etc., less than 500 μm, usually between about 0.1 μm and about 500 μm (e.g., 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, and / or 480 μm). In the devices used in the present disclosure, the microfluidic channels or chambers can have at least one cross-sectional dimension of about 10 μm to about 500 μm, or about 50 μm to about 500 μm.

[0047] In some embodiments, the microchannel cartridge further has a waste chamber provided downstream of the cell collection chamber and in fluid communication with a second flow path.

[0048] In some embodiments, the microchannel cartridge further has a third flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the waste chamber. In some embodiments, the width of the third flow path is greater than the width of the first flow path or the second flow path.

[0049] In some embodiments, the microchannel cartridge further includes a magnetic member disposed at or near its inlet or in the cell collection chamber, the magnetic member being configured to apply a magnetic force to the fluid sample received from the inlet. In some embodiments, the magnetic member may be disposed at or near the cell collection chamber. In some embodiments, the magnetic member may be disposed directly outside the second layer and at or near the cell collection chamber to facilitate capture of magnetically labeled cells within the cell collection chamber.

[0050] In some embodiments, the magnetic member can include magnetic beads, magnetic particles, magnetic strips, etc. In some embodiments, the magnetic member can have any three-dimensional shape as long as it is arranged to apply a magnetic force to a small concentrated region within the microchannel. The orientation of the magnetic member is configured to hold cells or cell fragments in a small concentrated region within the microchannel, thereby facilitating gravity separation, washing, and / or imaging.

[0051] In some embodiments, the magnetic member has a shape of a cube, a rectangular parallelepiped, a cylinder, a cone, a sphere, or a triangular prism / quadrangular prism. In some embodiments, the magnetic member has a shape of a rectangular parallelepiped (e.g., a hexahedron, a rectangular parallelepiped having six faces). In some embodiments, the vertices of the rectangular parallelepiped magnet are arranged towards the cell collection chamber. In some embodiments, the vertices of the rectangular parallelepiped magnet are arranged below the cell collection chamber. In some embodiments, the magnetic member has a cube magnet or a magnet having a substantially cubic shape. In some embodiments, the vertices of the cube magnet are arranged towards the cell collection chamber. In some embodiments, the vertices of the cube magnet are arranged below the cell collection chamber.

[0052] A method for improving the separation of a blood sample in a microchannel includes pre-filling the microchannel with a pretreatment solution, which, according to this method, increases the effective separation trajectories that enable unnecessary cells or cell debris to move further downward by gravity in the microchannel without physically increasing the dimensions (e.g., length) of the microchannel.

[0053] In some embodiments, the microchannel cartridge further includes a pretreatment solution that occupies 50% (e.g., 50%, 60%, 70%, 80%, 90%, 100%) of the microchannel. In some embodiments, the pretreatment solution includes distilled water, deionized water, or a phosphate buffered saline (PBS) buffer. In some embodiments, the pretreatment solution includes a phosphate buffered saline (PBS) buffer. In some embodiments, the pretreatment solution may occupy approximately 80% of the microchannel, and the labeled blood sample may occupy the remaining 20% of the microchannel.

[0054] In some embodiments, the first layer and the second layer may include other polymer slides together with the slide glass. In some embodiments, the polymer slide may include a polymer selected from polymethyl methacrylate (PMMA), aromatic thermoplastic polyester (e.g., polyacrylic acid), polycarbonate (PC), polybutadiene (PBD), polydimethylsiloxane (PDMS), polyethylene (PE), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyoxymethylene plastic (POM / acetal), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), thermoplastic polyurethane (TPU), and mixtures of two or more thereof.

[0055] In some embodiments, the microfluidic channel layer may include a thermoplastic polymer film. In some embodiments, the microfluidic channel may include a polymer selected from ethylene vinyl acetate (EVA) polymer, acrylic, acrylonitrile butadiene styrene (ABS) polymer, aromatic thermoplastic polyester (e.g., polyacrylic acid), polycarbonate (PC), polydimethylsiloxane (PDMS), polyglycolic acid (PGA), polylactic acid (PLA), polystyrene (PS), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), thermoplastic elastomer (TPE), thermoplastic polyurethane (TPU), natural or synthetic rubber, and mixtures of two or more thereof.

[0056] In some embodiments, the microfluidic channel layer may include a pressure-sensitive double-sided tape having styrene-butadiene-styrene (SBS), styrene-isoprene-styrene (SIS), and polypropylene coated with an adhesive, or a combination thereof. Non-limiting examples of the pressure-sensitive adhesive may include acrylic pressure-sensitive adhesives, rubber-based pressure-sensitive adhesives, silicone-based pressure-sensitive adhesives, urethane-based pressure-sensitive adhesives, polyester-based pressure-sensitive adhesives, and polyvinyl ether-based pressure-sensitive adhesives.

[0057] In some embodiments, the microfluidic channel layer is bonded between the first layer and the second layer, for example, using thermal lamination or an adhesive. In some embodiments, the first layer, the microfluidic channel layer, and the second layer can be manufactured as an integrated unit or provided separately.

[0058] In some embodiments, the first layer and / or the second layer are configured to enable real-time detection of a sample within the cell collection chamber. In some embodiments, at least a portion of the first layer or the second layer can include a transparent material such as a glass-based material. In some embodiments, the first layer or the second layer is a glass layer. In some embodiments, the first layer or the second layer can include a microscope slide suitable for direct observation under a microscope or direct detection by detection methods such as (for example, Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), fluorescence microscopy, and magnetic resonance (MR)).

[0059] In some embodiments, the second flow path can include a serpentine flow path, a straight flow path or a substantially straight flow path, a zigzag flow path, or a combination thereof.

[0060] In some embodiments, the microchannel cartridge can further include a coating attached to at least a portion of the first flow path, the cell collection chamber, the waste chamber, and / or the second flow path. In some embodiments, the coating may include chitosan (for example, neutral chitosan, chitosan salts, chitosan derivatives), chitin, polymethyl methacrylate (PMMA), silicone, polystyrene (PS), polysaccharides (for example, nonionic, ionic, cross-linked polysaccharides), poly-D-lysine, streptavidin, collagen, polyurethane, epoxy, or a combination thereof.

[0061] In some embodiments, the coating can comprise one or more antibodies. In some embodiments, one or more antibodies are biotinylated. The biotin moiety on the antibody can be used to attach additional labels (such as quantum dots) or magnetic beads or magnetic particles. In some embodiments, one or more antibodies are associated with quantum dots. Quantum dot-labeled antibodies can be used to detect specific types of cells or cell fragments based on specific surface antigens on the cells or cell fragments. As used herein, the terms "quantum dot", "Q dot", or "QD" refer to nanocrystalline particles made from materials that are semiconductors or insulating materials in bulk and have tunable photophysical properties in the range from near ultraviolet (UV) to far infrared (IR), particularly in the visible range. In some embodiments, the term "quantum dot" includes semiconductor nanocrystals (SCNs) comprising transition metals (non-limiting examples include Cd and Zn), and anions of group 16 of the IUPAC periodic table (non-limiting examples include Se, S, Te, and O). In some embodiments, the quantum dots may include Quantum Dot 525, Quantum Dot 565, Quantum Dot 585, Quantum Dot 605, Quantum Dot 655, Quantum Dot 705, and Quantum Dot 800.

[0062] In some embodiments, one or more antibodies are magnetically labeled. Magnetically labeled antibodies can be used to facilitate the capture of target cells or cell fragments based on specific surface antigens on the cells or cell fragments.

[0063] In some embodiments, one or more antibodies can comprise antibodies that specifically bind to complement pathway components, complement activation products, cell-bound complement activation products (CB-CAP), or cell fragment-bound complement activation products (CFB-CAP).

[0064] As used herein, "complement pathway component" can include proteins of the classical complement pathway, alternative complement pathway, and lectin complement pathway, such as C1, C4, C2, C3, and their fragments, such as C4a, C4b, C2a, C2b, C4b, C2a, C3a, C3b, C4c, C4d, iC4b, C3d, C3i, C3dg. Also included can be C5, C5b, C6, C7, C8, C9, C1inh, MASP1, MASP2, MBL, MAC, CR1, DAF, MCP, C4 binding protein (C4BP), factor H, factor B, C3bB, factor D, Bb, Ba, C3bBb, properdin, C3bBb, CD59, C3aR, C5aR, C1qR, CR2, CR3, and CR4, as well as other complement pathway components, receptors, and ligands not specifically described herein.

[0065] As used herein, "complement activation product" refers to fragments of the "complement pathway components" listed in the above paragraph, namely C4a, C4b, C2a, C2b, C4bC2a, C3a, C3b, C4c, C4d, iC4b, C3d, C3i, iC3b, C3c, and C3dg.

[0066] As used herein, "cell-bound complement activation product" or "CB-CAP" refers to a combination of one or more complement activation products and blood cells (such as erythrocytes, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, platelets, etc.) to which the complement activation products are bound (but not limited thereto). In some embodiments, CB-CAP can include complement activation products selected from BC4d, TC4d, EC4d, PC4d, RC4d, GC4d, MC4d, and combinations thereof.

[0067] As used herein, "cell fragment-bound complement activation product" or "CFB-CAP" refers to a complement activation product attached to a cell fragment of a blood cell, such as (including but not limited to) a cell fragment of a red blood cell, reticulocyte, T lymphocyte, B lymphocyte, monocyte, granulocyte, eosinophil, basophil, or platelet. As used in the present disclosure, CFB-CAP is derived from proteins of the classical complement pathway, alternative complement pathway, and complement lectin pathway, such as C1, C4, C2, C3, and their fragments, such as C4a, C4b, C2a, C2b, C4b, C2a, C3a, C3b, C4c, C4d, iC4b, C3d, C3i, C3dg, including complement pathway components. Also, C5, C5b, C6, C7, C8, C9, C1inh, MASP1, MASP2, MBL, MAC, CR1, DAF, MCP, C4-binding protein (C4BP), factor H, factor B, C3bB, factor D, Bb, Ba, C3bBb, properdin, C3bBb, CD59, C3aR, C5aR, C1qR, CR2, CR3, and CR4, as well as other complement pathway components, receptors, and ligands not specifically described herein can also be included. CFB-CAP can attach to cell fragments contained in cell lysates of cells such as blood cells (including but not limited to) red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, or basophils. In some embodiments, CFB-CAP attaches to at least one fragment (such as a cell fragment) of a red blood cell, lymphocyte, reticulocyte, platelet, granulocyte, monocyte, eosinophil, or basophil.

[0068] In some embodiments, one or more antibodies can include antibodies that specifically bind to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268.

[0069] In some embodiments, the first flow channel or the second flow channel has a height of about 50 μm to about 500 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, and / or 500 μm).

[0070] In some embodiments, the first flow channel or the second flow channel has a width of about 2 mm to about 20 mm (e.g., about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, and / or about 20 mm).

[0071] In some embodiments, the microchannel has a length of about 25 mm to about 75 mm (e.g., about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, and / or about 75 mm).

[0072] In some embodiments, the width of the microchannel cartridge may be about 3 mm to about 15 mm (e.g., about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, and / or about 15 mm).

[0073] In some embodiments, the cell collection chamber has a rectangular, elliptical, rhombic, circular, semi-circular, triangular, square, rectangular, pentagonal, or hexagonal shape. In some embodiments, the cell collection chamber has a rhombic shape.

[0074] In some embodiments, the cell collection chamber is about 9 mm 2 ~about 225 mm 2 (e.g., about 9 mm 2 , 30 mm 2 , 60 mm 2, 90 mm 2 , 120 mm 2 , 150 mm 2 , 180 mm 2 , 200 mm 2 , and / or 225 mm 2 has an area of

[0075] In some embodiments, the inlet or outlet may include an absorbent material disposed therein. In some embodiments, the absorbent material has a pore size in the range of about 100 μm to about 500 μm (e.g., 100 μm, 110 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, and / or 500 μm).

[0076] In some embodiments, the absorbent material can include absorbent fibers or sponges. In some embodiments, the absorbent material may include cotton, polyester, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), or combinations thereof.

[0077] In some embodiments, the absorbent material is configured to generate a capillary force greater than that of the second flow path to prevent air bubbles from entering the inlet.

[0078] In some embodiments, the microchannel cartridge has a wet filter paper in fluid communication with the outlet.

[0079] In some embodiments, the fluid sample can include whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, plasma, or combinations thereof. In some embodiments, the fluid sample can include blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets. In some embodiments, the fluid sample may include magnetically labeled cells.

[0080] In some embodiments, the microchannel cartridge of the present disclosure can be housed within a housing. In some embodiments, the housing may be a cassette. In some embodiments, the housing may include an opening for accessing the inlet of the microchannel cartridge.

[0081] In yet another aspect, the present disclosure further provides a microchannel cell separation device. The microchannel cell separation device can include one or more of the microchannel cartridges described herein and a rotating member configured to change the angle of the microchannel cartridge, whereby the flow rate of the fluid sample within the microchannel is adjusted.

[0082] In some embodiments, the rotating member can include a holding device for the microchannel cartridge, and the microchannel cartridge is removably attached to the holding device. In some embodiments, the rotating member can include an angle indicator having one or more marks indicating a loading position, a standby position, and / or a sorting position. In some embodiments, the rotating member is configured to rotate the microchannel cartridge continuously or intermittently to control the flow rate of the fluid sample. In some embodiments, the rotating member is driven by a motor.

[0083] In some embodiments, the microchannel cell separation device can further include a base for supporting the rotating member. In some embodiments, the microchannel cell separation device can further include one or more compression springs and / or one or more washers disposed between the base and the rotating member.

[0084] In another aspect, the present disclosure also provides a kit including one or more of the microchannel cartridges described herein. In some embodiments, the kit can further include an immunoassay reagent. In some embodiments, the immunoassay reagent can include an antibody. The antibody may be labeled with a quantum dot or a magnetic label.

[0085] In some embodiments, the kit can also include one or more additional reagents housed in the same container or a different container as the immunoassay reagent. For example, the kit can include a pretreatment solution or a washing solution provided in a container or a separate compartment different from the additional reagent.

[0086] In some embodiments, the kit can optionally include a device for collecting a sample (e.g., a biological sample). In some embodiments, the device for collecting the sample can include a capillary, a pipette, a syringe, a needle, a pump, and a swab. In some embodiments, the kit can include information materials. The information materials may be explanatory materials, instructional materials, marketing materials, or other materials related to the devices described herein and / or their methods of use.

[0087] Next, referring to FIG. 1, an exemplary process for manufacturing the microchannel cartridge 20 is shown. The microchannel cartridge 20 can be manufactured by laminating a microfluidic channel layer 22 between a first layer 21 and a second layer 23. The first layer 21 and / or the second layer 23 may be a slide glass. The microfluidic channel layer 22 can be joined to the first layer 21 and the second layer 23 by thermal lamination or an adhesive depending on its material. For example, at least a part of the microfluidic channel layer 22 can include a polymer adhesive sheet such as ethylene vinyl acetate (EVA) disposed between the first layer 21 and the second layer 23. A weight 24 can be used to enhance the adhesiveness between the first and second layers (21 and 23) and the microfluidic channel layer 22.

[0088] The first layer 21 or the second layer 23 can include (or be formed from) a polymer selected from polymethyl methacrylate, aromatic thermoplastic polyester (e.g., polyacrylic acid), polycarbonate (PC), polybutadiene (PBD), polydimethylsiloxane (PDMS), polyethylene (PE), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyoxymethylene plastic (POM / acetal), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), thermoplastic polyurethane (TPU), and a mixture of two or more thereof.

[0089] In addition to the above exemplary thermoplastic polymers, a predetermined pressure-sensitive material (e.g., in the form of a tape) can be used for the microfluidic channel layer 22. For example, these tapes may be styrene-butadiene-styrene (SBS), styrene-isoprene-styrene (SIS), or polypropylene coated with a pressure-sensitive adhesive. The pressure-sensitive adhesive can be selected from acrylic pressure-sensitive adhesives, rubber pressure-sensitive adhesives, silicone pressure-sensitive adhesives, urethane pressure-sensitive adhesives, polyester pressure-sensitive adhesives, and polyvinyl ether pressure-sensitive adhesives.

[0090] The inner surface of the microchannel 25 can be coated with a coating material such as chitosan, chitin, polymethyl methacrylate (PMMA), silicone, polystyrene (PS), polysaccharide, poly-D-lysine, streptavidin, collagen, polyurethane, epoxy, or a combination thereof, whereby the affinity of the antibody for the coated surface is improved.

[0091] Figure 2 shows various examples of a microscopic microfluidic channel of a straight flow path (as shown in the microchannel cartridge 40). In the microchannel cartridge 60, the straight flow path includes a first flow path 64 from the inlet 63 to the rhombic cell collection chamber 65 and a second straight flow path 67 leading to the outlet 62. In a first variant of the switchback channel, as shown in the microchannel cartridge 80, the first flow path 84 leads from the inlet 83 to the rhombic cell collection chamber 85, followed by an air bubble inhibiting switchback path 87 that functions as a second flow path leading to the waste chamber (i.e., red blood cell (RBC) collection chamber) 86 and the outlet 82. In the switchback channel cartridge 100, which is a second variant, the first flow path 104 leads from the inlet 103 to the elliptical cell collection chamber 105, followed by an air bubble inhibiting switchback path 107 that functions as a second flow path leading to the waste chamber (i.e., red blood cell (RBC) collection chamber) 106 and the outlet 102. In each case, the microscopic microfluidic channel includes an inlet 43, 63, 83, or 103 and an outlet 42, 62, 82, or 102. The cell collection chambers 45, 65, 85, or 105 may have any suitable shape such as rectangular, elliptical, rhombic, circular, semi-circular, triangular, square, rectangular, pentagonal, or hexagonal. For example, the cell collection chamber can be linear (45), rhombic (65, 85), or elliptical (105).

[0092] To facilitate the capture of magnetically labeled cells or cell fragments, a magnet 185 can be placed below the cell collection chambers 45, 65, 85, or 105. In the case of the microchannel cartridge 40, since there is no specific cell collection chamber, the magnet can be placed below the mark 180. The folded flow paths 87 or 107 are advantageous for preventing bubbles from moving within the channels during gravity separation when the inlet is above (e.g., 90 degrees above the horizontal line) and the outlet is below (e.g., -90 degrees below the horizontal line).

[0093] The overall external dimensions of a microchannel cartridge such as the microchannel cartridges 20, 40, 60, 80, or 100 may be from about 25 mm to about 75 mm (e.g., about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, and / or about 75 mm). The length of 75 mm is the standard length of a normal microscope slide. The width of the microchannel cartridge may be from about 3 mm to about 15 mm (e.g., about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, and / or about 15 mm).

[0094] The microchannel cartridges 40, 60, 80, or 100 may be three-dimensional. The height (or thickness) of the channels may be from about 50 μm to about 500 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, and / or 500 μm). The channel width may be from about 2 mm to about 20 mm (e.g., about 2 mm, about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, and / or about 20 mm).

[0095] The cell collection chambers 45, 65, 85, or 105 are about 9 mm 2 to about 225 mm 2 (e.g., about 9 mm 2 , 30 mm 2 , 60 mm 2 , 90 mm 2 , 120 mm 2 , 150 mm 2 , 180 mm 2 , 200 mm 2 , and / or 225 mm 2 ) and can have an area of

[0096] FIG. 3 shows that the inlet of the flow path can have a fine structure 121 or 141 such as a continuous bubble structure (open cell structure) made of cotton (as in the case of the microchannel cartridge C302), or a sponge-like closed cell structure (as in the case of the microchannel cartridge C303). These fine structures (121 or 141) inside the injection port increase the capillary force. As a result, since the capillary force at the inlet is greater than that at the outlet, when the inlet is above the horizontal line and the outlet is below the horizontal line, air intrusion can be prevented. The inlet of the microchannel is a capillary, and the fluid sample is aspirated by the capillary force. However, this capillary force is not sufficient to prevent air from entering the inlet during the cell separation process by gravity when the inlet is about 90 degrees above the horizontal line and the outlet is -90 degrees below the horizontal line. Therefore, a stronger capillary force is required at the inlet. By adding a plurality of fine structures inside the inlet, the capillary force at the inlet is enhanced. In other words, when the inlet is above and the outlet is below, the capillary force at the inlet must be greater than that of other microchannel regions including the outlet. For cell sorting by gravity, having an asymmetric capillary force (the capillary force at the inlet is greater than the capillary force at the outlet) to hold the fluid at the inlet and prevent air from entering the inlet is unique and advantageous compared to existing devices and methods. Also, since a mechanical valve for preventing air intrusion or blocking of the outlet is not required, the cell separation process of the present disclosure is much simpler and cost-effective.

[0097] The smaller the pores of the microstructure 121 or 141, the stronger the capillary force. However, if the pore size is smaller than the cells, there is a possibility that biological particles will clog the pores. When the pores are large, the capillary force becomes weak, and after air flows into the inlet, it flows into the cell collection chamber. When air flows into the cell collection chamber, all cells including the target cells are removed, and ultimately cell separation deteriorates. An appropriate pore diameter is important for effectively separating cells or cell fragments and may be in the range of about 100 μm to about 500 μm (e.g., 100 μm, 110 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, and / or 500 μm).

[0098] Two types of porous structures, a continuous bubble structure and an independent bubble structure, can be used. For example, fiber materials such as cotton fibers have a continuous bubble structure because adjacent cotton fibers are not physically connected to each other. The fiber material may include cotton, polyester, nylon, or combinations thereof. A sponge is a suitable example of an independent bubble structure because the cell walls are physically connected. The material of the sponge may be polyurethane, polyether, polyester, or combinations thereof. In either case, the pores need to allow both cells and liquid to pass through freely. Also, during the separation process by gravity and magnetism, it is necessary to have an appropriate (the stronger the better) capillary force that can hold the injected sample and all other fluids such as the pretreatment solution and the washing solution (e.g., phosphate buffered saline (PBS)).

[0099] In some embodiments, when the inlet of the microchannel cartridge is disposed below the outlet, the microchannel cartridge is configured to allow the fluid sample to be loaded into the inlet by gravity by delaying the passage of the fluid sample into the second flow path, and when disposed higher, to separate cells or cell fragments in the fluid sample by gravity by facilitating the passage of unwanted cells or cell fragments into the second flow path.

[0100] In some embodiments, the microchannel cartridge further includes a magnetic member disposed near the cell collection chamber. The magnetic member is configured to apply a magnetic force to the fluid sample received from the inlet.

[0101] FIG. 4 shows another exemplary configuration of the microchannel cartridge 401 of the present disclosure. The inside of the cell collection chamber includes a first coating 401, such as a chitosan coating, on top of a glass or other suitable substrate 402. The microchannel can further include a second coating 403 that includes one or more antibodies, such as an anti-CD19 antibody or an anti-CD20 antibody, on top of the first coating 401. A magnet 185 can be disposed below the cell collection chamber to facilitate capture of magnetically labeled cells or cell fragments.

[0102] Figure 5 shows an application example of the microchannel cartridge of the present disclosure for selectively capturing C4d-positive B cells. In this application, C4d-positive B cells are separated from other C4d-positive cells such as C4d-positive T cells, C4d-positive red blood cells (RBCs), and C4d-positive platelets. To selectively capture C4d-positive B cells, in step 501, a magnetic-labeled anti-CD19 antibody is added to the fluid sample. In step 503, when the magnetic-labeled anti-CD19 antibody specifically binds to surface CD19 expressed on B cells, the B cells are magnetically labeled. The magnetically labeled B cells can be captured in the cell collection chamber of the microchannel cartridge using a magnet 185 disposed near or behind the second layer of the microchannel cartridge. In step 505, other C4d-positive cells such as C4d-positive T cells, C4d-positive red blood cells (RBCs), and C4d-positive platelets can be washed away with a washing solution. Further, the selective capture state of C4d-positive B cells can be detected and monitored using a fluorescein isothiocyanate (FITC)-labeled anti-CD20 antibody.

[0103] Figure 6 shows an exemplary process of cell separation using the capillary microchannel cartridge, gravity, and magnet described herein. In step 110, the microchannel cartridge is not loaded and a bar magnet 185 is disposed behind the cell collection chamber 85. In step 111, using the capillary phenomenon of the microchannel, the pretreatment solution 160 is at least partially loaded into the cell collection chamber of the microchannel cartridge from the inlet. The pretreatment solution 160 can be added before loading the sample. The pretreatment solution 160 may be distilled water, deionized water, or a phosphate-buffered saline (PBS) buffer. The pretreatment solution 160 has at least two functions: (a) cleaning the inside of the cell collection chamber to remove debris, etc., and (b) increasing the weight when loading the sample. The pretreatment solution 160 is useful for avoiding a sudden increase in flow rate when injecting the sample. If the weight of the sample is light and the capillary force at the inlet is much greater than the weight of the sample, a sudden increase in flow rate may occur.

[0104] In step 112, the cell collection chamber of the microchannel cartridge is at least partially loaded with the blood sample 161. As a result, when the blood sample 161 is added, due to capillary action, the pretreatment solution 160 moves upward in the red blood cell (RBC) collection chamber and finally moves to the second flow path. The loading angle of the sample is an angle between -90 degrees below the horizontal line (the inlet is below and the outlet is above) and about 0 degrees. The user can select an angle between -90 degrees and 0 degrees (horizontal) according to the viscosity of the sample. For example, the least viscous sample can be injected at -90 degrees, and a very high-viscosity sample can be injected at 0 degrees, which is the horizontal line.

[0105] In step 113, after the addition of the blood sample 161, the user rotates the microchannel cartridge from the loading position to the standby position. The microchannel cartridge 20 is in a substantially horizontal position (i.e., about 0 degrees with respect to the ground or the horizontal line) for incubation (i.e., performing the first gravitational separation). This incubation time may range from about 1 minute to about 20 minutes.

[0106] In step 114, after the incubation period is completed, the microchannel cartridge is further rotated by about 90 degrees (i.e., in the opposite direction to the orientation of the device in steps 110 - 112), and gravitational separation (i.e., the second gravitational separation) is performed. This step may take about 5 minutes to about 20 minutes.

[0107] In step 115, heavy cells such as red blood cells (RBCs) begin to move downward due to gravity, while the magnetically labeled target cells are captured by the magnet 185 disposed behind or near the cell collection chamber.

[0108] In step 116, after magnetic separation, the microchannel cartridge 20 is returned to a substantially horizontal (i.e., substantially parallel to the ground) standby position, and by gradually adding the unused phosphate-buffered saline (PBS), unnecessary substances in the fluid sample such as unbound antibodies and fluorescent dyes can be removed.

[0109] As described above, gravity plays an important role in both the sample loading stage and the cell separation stage. First, during the loading of the sample, the flow rate can be reduced by gravity. When the inlet descends below the outlet, fluid flow of the fluid sample occurs due to capillary action. Although the flow path is mainly driven by capillary force, on the other hand, gravity can be utilized to decelerate (or control) the flow due to capillary action. Since the capillary force generated by the microchannel between the first layer and the second layer is strong, the injected sample moves at high speed and passes through the cell collection chamber. Due to such a fluid velocity, the possibility that target cells are captured by the cell collection chamber is very low. Therefore, it is essential to reduce the flow rate by gravity. Second, in the cell separation stage, gravity can be utilized to accelerate the passage of unnecessary components (such as unnecessary cells) in the fluid sample into the second flow path (or waste chamber), thereby increasing the efficiency of cell separation. During this gravity-based cell separation process (where the inlet is above and the outlet is below), the fluid does not move downward, and only the heavier cells continuously move downward due to gravity.

[0110] Next, referring to FIG. 7, a cell sorting device 30 that combines capillary, gravity, and magnetism is provided. This cell sorting device 30 includes a microchannel cartridge 20, a microchannel holding device 180 for the microchannel cartridge 20, a gravity control wheel 200, an indicator window 280, and two knobs 220 and 240 for right-handed and left-handed use. By rotating the gravity control wheel 200, the gravity of the fluid in the capillary microchannel cartridge 20 is controlled, thereby controlling the flow rate of the fluid in the flow path. The gravity control wheel 200 is connected to the microchannel holding device 180.

[0111] The gravity control wheel 200 is connected to the knob 220 so as to be rotatable manually. Further, the microchannel holding device 180 is connected to the second knob 240. The user can rotate the gravity control wheel 200 and the microchannel holding device 180 using both the knobs 220 and 240 to adjust their angles. The four components including the knobs 220 and 240, the gravity control wheel 200, and the microchannel holding device 180 are all connected via the rods 260a and 260b and are configured to rotate simultaneously. The microchannel holding device 180 can be made of any suitable material such as polycarbonate (e.g., transparent polycarbonate) or acrylic plastic. The microchannel holding device 180 is supported by the base 340 and the support bases 300a and 300b.

[0112] FIG. 8 shows the microchannel holding device 180 including the magnet 185, the support portion 182, and the holding portions 181 and 184 for the elastic band 183 such as a rubber band. The user can easily attach and remove the microchannel cartridge 20 by attaching or removing the elastic band 183 that fixes the microchannel cartridge 20 at a predetermined position within the system. With this design of the channel holding device, the microchannel cartridge 20 can be brought as close as possible to the magnet 185. The narrower the gap between the microchannel cartridge 20 and the magnet 185, the stronger the magnetic field for separating magnetically labeled target cells.

[0113] FIG. 9 shows the gravity control wheel 200 for controlling the angle of the microchannel holding device 180. The wheel has at least three main angles such as sample loading, standby, and gravity separation (sorting). Further, there are small units between sample loading and standby, and these units are useful for the user to appropriately rotate the gravity control wheel 200 while observing the actual flow rate. As shown in FIG. 7, the user can monitor the states of the channels such as loading, standby, gravity separation, and units using the indicator window 280 and the scale line 281.

[0114] FIG. 10 shows a side view of an exemplary cell sorting apparatus 300 that combines a capillary, gravity, and magnetism. Knobs 220 and 240 are connected to a gravity control wheel 200 via support bases 300a and 300b. Two washers 261 and 263 and a compression spring 262 can be arranged between knob 220 and support base 300a. By using more washers (e.g., 264, 266, 267, 269, 270, and 272) and / or more compression springs (e.g., 265, 268, and 271) at each joint of the rotating part, the angle of the separation device can be rotated smoothly.

[0115] FIG. 11 shows a cell separation apparatus having a detachable microchannel holding device 360. FIG. 12 shows the assembled cell separation apparatus including the detachable microchannel holding device 360 attached at a predetermined position. The detachable microchannel holding device 360 can be held in a predetermined position by a pair of positioning devices (i.e., 190 and 192)) that connect both sides of the holding device 360. The detachable channel holding device 360 can be fixed in a predetermined position by an attachment member 191 and a fastening member 193. In this configuration, the detachable channel holding device 360 can hold magnets of different sizes according to various requirements for cell separation. Detailed views are shown in FIGS. 13A - 13C. FIGS. 13B and 13C show a mode in which panels 364 and 366 move back and forth to accommodate magnets 370 of various sizes. The user can control panels 364 and 366 by using two knobs 361 and 367. Metal guide rails 362 and 368 are used for both the non - movable panel 363 and the movable magnet holders 364 and 366. Two rubber band holders 365 and 369 hold one or more rubber bands. The rubber band 183 can be attached using the rubber band holders 365 and 369.

[0116] As shown in FIG. 14, the cell sorting device 300 of the present disclosure can be implemented as an automated system monitored by a mobile device such as a smartphone 380. For example, this system can be connected to a smartphone 380 that displays cell images and cell counts via a wireless or wired connection. Further, the control device 382 can rotate the knob 381 via the belt 383, for example, based on an algorithm that uses loading, standby, and gravity separation. The control device 382 can communicate with the cell sorting device 30 via the power transmission system 384. Additionally and / or optionally, the control device 382 can also supply power to the cell sorting device 30 via the power transmission system 384. Target cells or cell fragments in the cell collection chamber can be detected by appropriate detection methods such as Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), fluorescence microscopy, and magnetic resonance (MR).

[0117] FIG. 15 shows a small fully automated system 400 having a protective cover and a user interface 401. Optionally, a manual knob 80 and a gravity control wheel 200 may be added to this small design in case the user desires a manual operation of the cell separation device.

[0118] FIG. 16 shows a manually operated cell separation device 500 that can rotate left and right. In this configuration, the user can visually observe the back side of the microchannel cartridge 20 and directly observe the cell separation process. The manually operated cell separation device 500 can be rotated by rotating the upper circular panel 340 with respect to the lower circular panel 440. There is a ball bearing 442 between the upper panel 440 and the lower panel 441 to minimize friction. Although the configuration of FIG. 7 is used for illustration in this embodiment, the panels and functions shown in FIG. 16 can also be included in the configurations of other systems such as FIGS. 11 - 12 or FIG. 17 (below).

[0119] An example of a gravity cell separation device equipped with a cubic magnet is shown in Fig. 17. In this embodiment, the holding device is composed of a bar 400, an upper panel 405, and a lower panel 406 instead of an elastic band. The bar 400 is used to fix the microchannel on the upper panel 405. Both the bar 400 and the upper panel 405 are coated with silicone to obtain good traction, thereby completely preventing the movement of the microchannel cartridge. The lower panel 406 houses the cubic magnet. The lower panel 406 rotates when the user turns the knob 80 or 81. The bar 400 is connected to the upper panel 405 via a hinge 401. The upper panel 405 is connected to the lower panel 406 via a hinge 402. The cubic magnet 404 is attached to the lower panel 406 and contacts the microchannel through the rectangular space of the upper panel 405. The lock systems 407 and 408 lock the upper panel 405 and the lower panel 406 so that they are held integrally during the gravity separation process. The lock system 407 maintains good contact between the microchannel and the apex of the cubic magnet 404.

[0120] Since one side of the bar 400 is connected to the upper panel 405 via the hinge 401, it can move up and down. The hinge 401 is fixed to the upper panel 405 by a fixing member 401. The other end of the bar 400 can be removably held toward the upper panel 406 by a fixing member 403, so that the microchannel cartridge can be clamped on the upper panel 406 during sample loading and separation. The upper panel 405 is sequentially connected to the lower panel 406 by a hinge 402 so that it can move away from the lower panel 406 in a stable state. During sample loading and separation, the upper panel 405 and the lower panel 406 are held together by a locking mechanism including, for example, a knob 407 and a latch 408. During cell separation by gravity or washing with phosphate buffered saline (PBS), the upper panel 405 or the lower panel 406 can be stopped by a stopper 409.

[0121] Figure 18 shows a side view of the holding device of Figure 17. The bar 400 and the panels 405 and 406 move or rotate independently. In Figure 17, the bar 400 is in an open state to receive the microchannel cartridge. Figure 19 shows that the microchannel cartridge is disposed between the bar 400 and the panel 405 and locked between the bar 400 and the panel 405.

[0122] As shown in Figure 19, after the bar 400 is closed, the microchannel cartridge is disposed substantially horizontally for, for example, 25 minutes. This step is called the first gravity separation. In this step, all cells precipitate to the bottom of the microchannel cartridge. During this step, a portion of the B cells moves to the apex of the cubic magnet. Figure 21 shows a second gravity separation step in which the cartridge is rotated 90 degrees and all cells start to move towards the outlet of the cartridge by gravity. However, the B cells bound to the magnetic nanoparticles are attracted to the apex of the cubic magnet, forming an island of concentrated B cells. After the separation step is performed at the position of Figure 20, the holding device is returned to the position of Figure 19 for washing with phosphate buffered saline (PBS) after gravity separation. Figure 21 shows a state in which the panel 405 is separated from the panel 406, whereby the microchannel cartridge 20 can be removed from the panel 405. The microchannel cartridge 20 is removed from the holding device after the washing step with phosphate buffered saline (PBS).

[0123] Method for separating and detecting cells or cell fragments In another aspect, the present disclosure further provides a method for separating and detecting cells or cell fragments in a fluid sample. In some embodiments, the method comprises: (a) disposing the microchannel cartridge described herein at a sample loading angle; (b) introducing a fluid sample into the microchannel through an inlet of the microchannel; (c) performing a first gravitational separation by incubating the fluid sample for a first period; and (d) disposing the microchannel cartridge at a sorting angle for gravitational separation for a second period to perform a second gravitational separation, wherein the flow of the target cells or cell fragments is induced into a cell collection chamber by this step.

[0124] In some embodiments, the first period is from about 1 minute to about 45 minutes (e.g., in each case, about 1 minute, about 3 minutes, about 5 minutes, about 7 minutes, about 9 minutes, about 11 minutes, about 13 minutes, about 15 minutes, about 17 minutes, about 19 minutes, about 21 minutes, about 23 minutes, about 25 minutes, about 27 minutes, about 29 minutes, about 31 minutes, about 33 minutes, about 35 minutes, about 37 minutes, about 39 minutes, about 41 minutes, about 43 minutes, and / or about 45 minutes). In some embodiments, the second period is from about 10 minutes to about 120 minutes (e.g., in each case, about 10 minutes, about 12 minutes, about 14 minutes, about 16 minutes, about 18 minutes, about 20 minutes, about 22 minutes, about 24 minutes, about 26 minutes, about 28 minutes, about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 42 minutes, about 44 minutes, about 46 minutes, about 48 minutes, about 50 minutes, about 52 minutes, about 54 minutes, about 56 minutes, about 58 minutes, about 60 minutes, about 62 minutes, about 64 minutes, about 66 minutes, about 68 minutes, about 70 minutes, about 72 minutes, about 74 minutes, about 76 minutes, about 78 minutes, about 80 minutes, about 82 minutes, about 84 minutes, about 86 minutes, about 88 minutes, about 90 minutes, about 92 minutes, about 94 minutes, about 96 minutes, about 98 minutes, about 100 minutes, about 102 minutes, about 104 minutes, about 106 minutes, about 108 minutes, about 110 minutes, about 112 minutes, about 114 minutes, about 116 minutes, about 118 minutes, and / or about 120 minutes).

[0125] In some embodiments, the loading angle is from about -90 degrees to about 0 degrees below the horizontal line (e.g., about -90 degrees, about -85 degrees, about -80 degrees, about -75 degrees, about -70 degrees, about -65 degrees, about -60 degrees, about -55 degrees, about -50 degrees, about -45 degrees, about -40 degrees, about -30 degrees, about -25 degrees, about -20 degrees, about -15 degrees, about -10 degrees, about -5 degrees, or about 0 degrees below the horizontal line).

[0126] In some embodiments, the sorting angle is from about 90 degrees to about 0 degrees above the horizontal line (e.g., about 90 degrees, about 85 degrees, about 80 degrees, about 75 degrees, about 70 degrees, about 65 degrees, about 60 degrees, about 55 degrees, about 50 degrees, about 45 degrees, about 40 degrees, about 30 degrees, about 25 degrees, about 20 degrees, about 15 degrees, about 10 degrees, about 5 degrees, or about 0 degrees above the horizontal line).

[0127] In some embodiments, the method can further include the step of adding an immunological reagent before the step of introducing the fluid sample.

[0128] In some embodiments, the immunological reagent can include one or more antibodies. In some embodiments, the one or more antibodies may be biotinylated. In some embodiments, the one or more antibodies may be labeled with quantum dots or magnetic particles.

[0129] In some embodiments, the one or more antibodies can include antibodies that specifically bind to complement pathway components, complement activation products, cell-bound complement activation products (CB-CAP), or cell fragment-bound complement activation products (CFB-CAP).

[0130] In some embodiments, the one or more antibodies can include antibodies that specifically bind to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268.

[0131] In some embodiments, the fluid sample can include whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, plasma, or combinations thereof. In some embodiments, the fluid sample can include blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets.

[0132] In some embodiments, the fluid sample may include magnetically labeled cells.

[0133] As used herein, "sample" includes a sample containing a biological substance. The sample may be, for example, a fluid sample (e.g., a blood sample). The sample may be a part of a larger sample. In some embodiments, the fluid sample may include a biological fluid such as blood (e.g., whole blood), plasma, sputum, urine, sweat, urine swab, semen, saliva, buccal swab, or combinations thereof. The sample may be a forensic sample. As used herein, "sample", "body fluid sample", "fluid sample", "individual sample", "subject sample", or "patient sample", etc. in the sense of obtaining a sample from a patient, subject or individual refers to a sample containing plasma, serum, whole blood, cerebrospinal fluid (CSF), urine, saliva, tears, semen, colostrum, or any recoverable body fluid in one or more of the various assays disclosed herein.

[0134] The sample may be pretreated before being introduced into the system. In some embodiments, the pretreatment can include extraction from substances that are not compatible with the system, quantification of the amount of cells, DNA, RNA, or other biopolymers or molecules, concentration of the sample, separation of cell types (such as separation of sperm from epithelial cells), bead treatment, other concentration methods, or other manipulations of the sample.

[0135] The amount of fluid sample applied to the inlet of the microchannel cartridge may vary as long as it is sufficient to provide the desired capillary flow and assay operability. In some embodiments, the devices of the present disclosure are compatible with small amounts of sample (e.g., 0.5 μL to 50 μL).

[0136] The sample can be applied to the sample application area using any convenient protocol, for example, by means of a dropper, pipette, syringe, etc. Further, the fluid sample can be applied to the sample receiving area together with any suitable liquid, such as a buffer. Non-limiting examples of any suitable liquid can include, but are not limited to, buffers, cell culture media (e.g., DMEM), etc. Non-limiting examples of buffers can include, but are not limited to, Tris, Tricine, MOPS, HEPES, PIPES, MES, PBS, TBS, etc. In one example, the suitable liquid can be mixed with the fluid sample before being applied to the microchannel. In another example, the suitable liquid can be applied to the microchannel simultaneously with, before, or after the application of the fluid sample.

[0137] In some embodiments, the method may include loading a pretreatment solution into the microchannel before the step of introducing the fluid sample. In some embodiments, the pretreatment solution is selected from distilled water, deionized water, and phosphate buffered saline.

[0138] In some embodiments, the method may include loading a washing solution into the microchannel to wash cells or cell debris after the step of introducing the fluid sample. In some embodiments, the washing solution includes phosphate buffered saline.

[0139] In some embodiments, the method can further include detecting cells or cell fragments within the cell collection chamber by an appropriate detection method such as Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), fluorescence microscopy, and magnetic resonance (MR). In some embodiments, the step of detecting may include the step of counting the number of cells.

[0140] In some embodiments, the method can include comparing the determined level of the captured cells or cell fragments with a control level of the captured cells or cell fragments. In some embodiments, the method can further include, when the determined level is elevated compared to the control level, identifying that the subject has a disease or disorder.

[0141] In some embodiments, the method further has the step of disposing a wet absorbent material at the outlet after washing the cells or cell fragments, the wet absorbent material being in fluid communication with the outlet and facilitating the removal of unwanted cells or cell fragments. In some embodiments, the wet absorbent material has a wet filter paper. In some embodiments, the method further has the step of applying an air stream (e.g., room temperature air or hot air) to the wet absorbent material to gradually evaporate the liquid from the wet absorbent material to facilitate the removal of unwanted cells or cell fragments.

[0142] In some embodiments, the method further has the step of applying hot air to the outlet of the microchannel cartridge to facilitate the removal of the dried coagulated blood waste.

[0143] As used herein, in some embodiments, the "control" level refers to the level of captured cells or cell fragments taken from samples taken from one or more individuals not suffering from the disease or disorder under investigation. This level can be measured for each individual or based on aggregated values such as an average value. Also, the "control" level can be determined by analyzing a population of individuals who have the disease or disorder but have not experienced the acute phase of the disease or disorder. A "control" sample is used to obtain the above "control" level. A "control" sample can be taken from one or more individuals not suffering from the disease or disorder under investigation. Also, a "control" sample can be taken from a population of individuals who have the disease or disorder but have not experienced the acute phase of the disease or disorder. In some embodiments, the "control" level is the level obtained from the same individual at different times, where the diagnosis has been made or the condition is being monitored. In some embodiments, the "control" level or "control" sample may refer to a level or sample obtained from the same patient at an earlier time, for example, weeks, months, or years ago.

[0144] As used herein, when it is said that "the determined level is elevated compared to the control level", it indicates a positive change from the value of the control level.

[0145] Other Definitions To facilitate understanding of the detailed description of the compositions and methods according to the present disclosure and to clearly disclose various aspects of the present disclosure, several explicit definitions are provided throughout the following and the entire detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0146] As used herein, the terms "capillary action" or "capillary force" refer to the forces arising from adhesive and surface tensions acting on a fluid within a small passage such as a tube or within a container, by which the fluid is moved through the container (such as a substrate or a capillary within a substrate). When the adhesive force resulting from the intermolecular attraction between the fluid molecules and the wall of the container in which the fluid is contained is stronger than the cohesive force within the fluid resulting from the intermolecular attraction between the fluid molecules, an upward force is generated on the fluid at the edge of the container. This force pulls the fluid at the edge of the container upward, creating a meniscus. At the same time, the surface tension resulting from the increase in the cohesive force between the fluid molecules at the surface of the fluid acts to hold the surface in place, causing not only the edge but also the entire surface of the fluid to move upward. The combination of these forces is called capillary force or capillary action. As used herein, the terms "wicking" or "wicking forces" refer to the movement of a fluid through a porous medium that occurs as a result of capillary forces occurring within the pores of the porous medium. Usually, in a porous medium, capillary forces are generated, for example, by the proximity of small-diameter pores or fibers, and a certain degree (to the extent that the fluid passes through the porous medium) of capillary action occurs.

[0147] As used herein, the term "wicking" refers to the movement of a fluid through a porous medium that occurs as a result of capillary forces occurring within the pores of the porous medium. Usually, in a porous medium, capillary forces are generated, for example, by the proximity of small-diameter pores or fibers, and a certain degree (to the extent that the fluid passes through the porous medium) of capillary action occurs. The term "wicking rate" refers to the movement of the fluid per unit time, i.e., the distance traveled by the fluid within a specific time period.

[0148] As used herein, the term "thin film" includes thin films and sheets of any shape, including rectangular, square, or other desired shapes. The thin films described herein may be of any desired thickness and size. For example, the thin film may have a relatively thin thickness of about 0.1 μm to about 1 mm. The thin film may be single-layer or multi-layer.

[0149] The terms "patient", "individual", and "subject" are used interchangeably and generally refer to any organism that utilizes the disclosed methodology to collect a body fluid sample in order to perform the diagnostic or monitoring methods described herein. The patient may be an animal such as a human. Also, the patient may be a domestic or farm animal. A "patient" or "individual" may sometimes be referred to as a subject.

[0150] As used herein, the term "disease" is generally synonymous with the terms "disorder" and "condition" (such as a medical condition) in that it impairs normal function, typically manifests as characteristic signs and symptoms, and reflects an abnormal state of the body or a part thereof of a human or animal, reducing the lifespan or quality of life of the human or animal, and is used interchangeably with these terms.

[0151] As used herein, "diagnosis" means identifying the presence or nature of a pathological condition such as systemic lupus erythematosus ("SLE"). Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the proportion of patients with the disease who are determined to be positive (the proportion of "true positives"). Patients with the disease who are not detected by the assay are "false negatives". Subjects who do not have the disease and are determined to be negative by the assay are called "true negatives". The "specificity" of a diagnostic assay is the value obtained by subtracting the false positive rate from 1, where the "false positive" rate is defined as the proportion of those without the disease who are determined to be positive. While a particular diagnostic method may not provide a definitive diagnosis of the condition, it may be sufficient if the method provides positive signs that are useful for diagnosis. The terms "relating to diagnosis", "diagnostic method", and "diagnosis" can be used interchangeably with "identifying", "identifying method", and "identifying".

[0152] As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and polyreactive antibodies), and antibody fragments. Thus, the term "antibody" as used in any context herein means any specific binding member, including (but not limited to) immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and biologically relevant fragments including (but not limited to) Fab, F(ab')2, Fv, and scFv (single-chain or related entities), or any specific binding member of such fragments. In the art, an antibody is understood to be a glycoprotein having at least two heavy chains (H chains) interconnected by disulfide bonds and two light chains (L chains), or the antigen-binding portion of such glycoprotein. The heavy chain is composed of a heavy-chain variable region (VH) and heavy-chain constant regions (CH1, CH2, CH3). The light chain is composed of a light-chain variable region (VL) and a light-chain constant region (CL). Both the variable regions of the heavy and light chains are composed of framework regions (FWR) and complementarity-determining regions (CDR). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2, CDR3) represent hypervariable regions and are arranged in the order of FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4 from the NH2 terminus towards the COOH terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens, and the constant regions mediate binding to host tissues or factors of immunoglobulins depending on the isotype.

[0153] As used herein, the definition of "antibody" also includes chimeric antibodies, humanized antibodies, recombinant antibodies, human antibodies generated from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those of skill in the art.

[0154] An "antibody fragment" has a portion of a complete antibody, such as the antigen-binding or variable region of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, diabodies, linear antibodies (see, e.g., U.S. Patent No. 5,641,870, and Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0155] As used herein, when the term "contact" is used in reference to any set of components, it includes any process by which the contacting components are combined in the same mixture (e.g., added to the same compartment or solution), but does not necessarily require actual physical contact between the recited components. The recited components can be contacted in any order or in any combination (or sub-combination), and can include situations where one or some of the recited components are removed from the mixture before any optionally recited additional components are added. For example, "contacting A with B and C" includes any and all of the following situations: (i) mixing A with C and then adding B to the mixture, (ii) mixing A and B in the mixture, removing B from the mixture, and then adding C to the mixture, and (iii) adding A to a mixture of B and C.

[0156] As used herein, the term "in vitro" refers to events that occur in an artificial environment such as in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0157] As used herein, the term "in vivo" refers to events that occur within a multicellular organism such as a non-human animal.

[0158] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0159] The terms "including", "comprising", "containing", or "having" and variations thereof mean, unless otherwise specified, the items listed next to the term and their equivalents, as well as additional subject matter.

[0160] Phrases such as "in one embodiment", "in various embodiments", "in some embodiments", etc. are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may refer to the same embodiment, except where the context indicates otherwise.

[0161] The term "and / or" or " / " means any one of the items associated with this term, any combination of the items, or all of the items.

[0162] The term "substantially" does not exclude "completely". For example, a composition in which Y is "substantially not included" may possibly not include Y completely. Optionally, the term "substantially" may be omitted from the definitions of the present disclosure.

[0163] As used herein, the terms "about" or "approximately" when applied to one or more target values refer to values similar to the recited reference values. In some embodiments, the terms "about" or "approximately" refer to a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than the reference value) from the recited reference value, unless otherwise specified or apparent from the context (except where the target numerical value exceeds 100% of the possible value). Unless otherwise specified herein, the term "about" is intended to include approximate values of the recited ranges that are equivalent with respect to the functionality of the individual components, compositions, or embodiments, e.g., weight percentages.

[0164] It should be understood that when values and ranges are provided herein, all values and sub-ranges subsumed within the recited values and ranges are meant to be encompassed within the scope of this disclosure. Further, all values within these ranges, as well as the upper or lower limits of these ranges of values, are contemplated in this application.

[0165] As used herein, the term "each" when used in relation to items of a collection is intended to identify individual items within the collection, but not necessarily to refer to every item within the collection. Exceptions may occur where it is clear from the explicit disclosure or context that the foregoing is not intended.

[0166] Any and all examples, or exemplary language (e.g., "such as") provided in this specification are used to more specifically illustrate the present invention and, unless otherwise indicated, do not limit the scope of the present disclosure. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention. As used herein, the term "exemplary" is intended to mean "by way of example" and is not intended to indicate that a particular exemplary item is preferred or essential.

[0167] All methods described in this specification, unless otherwise specified herein or clearly inconsistent with the context, are performed in any suitable order. For any of the methods provided, the steps of the method can be performed simultaneously or sequentially. When the steps of the method are performed sequentially, they can be performed in any order unless otherwise specified.

[0168] When a method has a combination of multiple steps, unless otherwise specifically described herein, each combination or sub-combination of steps, and all combinations or sub-combinations are included within the scope of the present disclosure.

[0169] Each publication, patent application, patent, and other reference cited in this specification is incorporated by reference in its entirety, to the extent not inconsistent with the present disclosure. The publications disclosed herein are provided only with respect to the disclosure content before the filing date of the present disclosure. No statement in this specification should be construed as an admission that the present invention has any right to antedate such disclosure by virtue of prior invention. Further, the provided publication dates may be different from the actual publication dates and may need to be independently verified in some cases.

[0170] The examples and embodiments described in the specification are for illustrative purposes only, and various modifications or changes are suggested to those skilled in the art in view of them. It is understood that the said examples and embodiments are included within the gist and scope of the present invention and within the appended claims.

[0171] Example

Example

[0172] Manufacture of Microchannel Cartridge and Cell Separation Device By laminating an ethylene vinyl acetate (EVA) polymer thin film between two standard glass microscope slides (the first layer and the second layer), a microchannel in a folded shape was manufactured. A commercially available ethylene vinyl acetate (EVA) sheet with a thickness of 164 μm was used. The folded shape on the ethylene vinyl acetate (EVA) sheet was cut with a razor before the thermal lamination process. Since the thickness shrinks during lamination, four EVA sheets (for example, dimensions 25 mm x 75 mm) were placed between two microscope slide glasses to manufacture a microchannel with a thickness of 350 μm. The ethylene vinyl acetate (EVA) sheet together with the two slide glasses was placed on an electric heater at 100°C. After performing the lamination process for 4 minutes, the laminated microchannel was taken out and immediately placed on a stainless steel table to lower the temperature.

[0173] An exemplary cell separation device that combines capillary, gravity, and magnetism was manufactured. The cell separation device has a holding device for the microchannel cartridge, and the microchannel cartridge can be easily attached to and removed from this holding device for sample loading, cell separation, and analysis. The dimensions of the exemplary cell separation device are 12 inches x 5 inches x 5 inches, and the weight is about 1 kg. The materials used in the manufacture of the cell separation device include wood, acrylate plastic, rubber bands, bar magnets, compression springs, and washers.

Example

[0174] Separation of B cells by capillary separation method only and by a separation method combining capillary and gravity The fabricated microchannel was placed on a cell separation device that combined capillary, gravity, and magnetism. The inlet was 90 degrees below the horizontal line, and 40 μl of phosphate-buffered saline (PBS) pretreatment solution was injected. The injected PBS solution gradually moved into the cell collection chamber as the angle of the inlet increased from -80 degrees, -70 degrees, to -60 degrees. After adding the pretreatment solution, 30 μl of labeled blood sample was injected, and then the angle of the inlet was increased to -80 degrees, -70 degrees, and -60 degrees. After injecting all the blood, the separation device was set to the standby position at 0 degrees with respect to the horizontal line, and the blood sample was incubated in the dark at this position for 5 minutes. After incubation, the separation device was rotated to +90 degrees with respect to the horizontal line for gravity separation. At this position, red blood cells (RBCs) moved downward toward the RBC collection chamber, while the target B cells were maintained in the upper magnet chamber. During this gravity separation, only biological particles such as cells and cell debris moved, and the fluid did not move. After gravity separation, the separation device was moved to the standby position at 0 degrees with respect to the horizontal line, and 80 μl of phosphate-buffered saline (PBS) was injected at this position to wash away unbound fluorescent dyes and other unbound biological particles.

[0175] For comparison, a control microchannel was used to separate B cells by capillary separation method only. Figure 22 shows the difference in exemplary measurements of B cells collected between a conventional microchannel using only capillary (without gravity) and an exemplary microchannel of the present disclosure using both capillary and gravity. In the microchannel without using gravity, only 6 B cells were collected. In contrast, 200 B cells, corresponding to a 33-fold enrichment of B cells, were captured in the microchannel of the present disclosure that combined capillary and gravity.

Example

[0176] Comparison of the separation method combining capillary and gravity and the separation of B cells by a mechanically pumped cell separation device Using a protocol similar to the one described in Example 2, the microchannel cartridge and method combining capillary and gravity of the present disclosure were compared with a mechanical pump-driven cell separation device. As shown in FIG. 23, the enrichment of B cells was significantly improved by the device and method of the present disclosure. The magnetic microchannel utilizing capillary and gravity can separate approximately 97 times more B cells than a mechanical pump-driven cell separation device.

[0177] In addition to the significant improvement in B cell enrichment, the microchannel cartridge and method combining capillary and gravity of the present disclosure achieved more uniform cell collection on the surface of the microchannel. FIG. 24A shows the cells collected in a microchannel driven by a mechanical pump, but the captured cells are not uniformly distributed throughout the microchannel. However, as shown in FIG. 24B, in the cell separation device utilizing capillary and gravity, a uniform monolayer of cells was obtained in the microchannel. Furthermore, this cell monolayer is denser than that of the mechanical pump-driven cell separation device. The fact that the monolayer is denser means a higher ability to collect more target cells (B cells in this example), which supports the results of FIG. 23.

Example

[0178] Labeling of whole blood for gravity channel separation / C4D detection on B cells A small aliquot of 10 μl of whole blood was diluted 1:1 using a magnetic separation buffer (BD IMag® buffer from BD Biosciences) containing BSA and EDTA in phosphate buffered saline (PBS). The IMAG buffer was formulated to reduce non-specific binding of antibodies. 1 μl of mouse anti-human CD19 magnetic particles, 3 μl of mouse anti-human CD20 FITC antibody (from BD Biosciences), and an anti-human C4d 9A10 biotin / streptavidin conjugate antibody (from BD Biosciences) at a nominal concentration (0.32 μg) were added to the diluted whole blood. The total volume of whole blood and antibodies before magnetic separation was only 33 μl.

[0179] The anti-human C4d 9A10 antibody was prepared by treating the antibody with a biotinylation kit (manufactured by Abcam) before use. The final concentration of the biotinylated anti-C4d antibody was 1 mg / ml. Streptavidin conjugated to Alexa 647 (manufactured by Thermo Fisher Scientific) was diluted to a concentration of 0.7 mg / ml before complexing with anti-C4d 9A10. Next, the biotinylated anti-C4d antibody was incubated with an equal volume of streptavidin conjugated to Alexa 647 in the dark at room temperature for 30 minutes. After incubation, the anti-C4d antibody was diluted at a ratio of 25 μl of phosphate-buffered saline (PBS) per 4 μg of the antibody. The diluted and complexed antibody was centrifuged at 20,000 g for 3 minutes to remove unbound and aggregated streptavidin that did not bind to Alexa 647. The centrifuged anti-C4d biotin / streptavidin Alexa 647 complex was removed from the supernatant and added to a new microcentrifuge tube. 2 μl of the complexed antibody was further diluted with 8 μl of magnetic separation buffer to a final working concentration of 0.32 μg. A total volume of 10 μl was used for the separation assay. Figure 27 is an example showing the difference in fluorescence signals between the anti-C4d9A10 antibody directly conjugated with Alexa 647 and the same antibody complexed with biotinylated streptavidin conjugated to Alexa 647. By using the biotin / streptavidin complex antibody, the C4d signal on the captured B cells can be amplified.

Example

[0180] Imaging and analysis of B cells separated by the gravity channel All images were taken at a magnification of 20x using an EVOS fluorescence microscope (Figures 24A - 24C). The GFP channel of the microscope was used to scan for anti-CD20 FITC-positive B cells that fluoresce in the surrounding area of the magnetically separated region. After detecting the B cells, three images were taken for each B cell: an image using the brightfield channel, an image using the GFP channel, and an image using the Cy5 channel. All images were taken in 16-bit tiff file format.

[0181] All image files were loaded into an image analysis plugin created for ImageJ software. This plugin measures all cells within the image plane using bright-field images and separates the cells based on size and circularity. To quantify the number and level of C4d on B cells, images were created by overlaying anti-CD20 FITC-labeled B cells imaged in the GFP channel and anti-C4d-labeled cells imaged in the Cy5 channel and combining them with bright-field images. This plugin performs cell measurement, fluorescence image overlay, fluorescence measurement of each pixel, and calculation of the background of both fluorescence channels at 2 - 3 seconds per image area. Also, this plugin generates a "cell stat" file containing statistical data for each region. The total cell statistic files for each image region were uploaded to the comprehensive statistic function in another tab of the plugin. This comprehensive statistic function compiles the cell statistic files into a single dataset for each patient sample. This dataset is then used to calculate the overall background of all images. This background can be used to set a threshold for determining which cells are B cells (anti-CD20 FITC positive) by entering the background data into a specific threshold field. By using this threshold, it becomes possible to accurately measure B cells by the plugin and determine the fluorescence signals of both anti-CD20 and anti-C4d.

Example

[0182] Analysis of Autofluorescence and Background In any assay, the background varies depending on the material in which the cells are retained and the efficiency of removal of unbound antibodies.

[0183] Table 1 shows a comparison of two materials used for separating B cells. One is a double slide glass used in the gravity channel, and the other is a cartridge manufactured for use with a fluid pump. The cartridge uses large acrylic pieces, which are required to manufacture an inlet for connecting to the pump and a waste container for holding the contents generated during the washing process. Images of various regions along the channels were taken 10 times with and without phosphate buffered saline (PBS) using both the Cy5 channel and the GFP channel, and these images were analyzed using the ImageJ plugin. As a result, it was shown that the background increased in both the Cy5 and GFP channels in the acrylic cartridge, and especially increased further in the GFP channel. The closer the captured images were to the adhesive used to form the actual channels through which the cells flowed in the separation assay, the more the GFP background of the acrylic cartridge increased. When the fluorescence signal of the GFP background was high, the signal produced by the anti-CD20 FITC B cell detection antibody was masked, and the detection of B cells by the image analysis plugin became inaccurate. As shown in FIGS. 28A to 28C, in the folded channel, since there is no need to use a pump, there is no need to create an area for the pump connection part and the waste collection tank, and it becomes possible to use a thin slide glass. By using a thin slide glass, the background autofluorescence of the materials used is reduced, and it becomes possible to detect B cells more accurately.

[0184]

Table 1

[0185] The following Tables 2 and 3 show the washing efficiency of the gravity-utilizing channels of the present disclosure when washing unbound antibodies, compared to the case of using a centrifuge. B cells were separated using a flow cytometry protocol, and the washed cells were dispersed on a slide and then imaged. As a result, it was shown that the gravity channel efficiently washes unbound antibodies from the cells. The C4d signal resulted in a higher value in the gravity-utilizing channels of the present disclosure, while the amount of phosphate-buffered saline (PBS) used was 80 μl, compared to 6 ml in the flow cytometry protocol. Also, based on the comparison results of the C4d signals (below) of the two assays, the background-to-signal ratio is sufficient to produce a strong correlation between the two methods. The numerical results in the following Tables 2 and 3 were measured as the average fluorescence signal per pixel.

[0186]

Table 2

[0187]

Table 3

Example

[0188] Correlation between Flow Cytometry Analysis and Image Analysis Using the method described above, the levels of C4d present in B cells of patients recruited at the Allegheny Health Network Autoimmunity Institute were determined. These results were compared to those obtained by flow cytometry. Flow cytometry requires much more blood, 50 μl, compared to the mere 10 μl of blood used in the gravity-based channel. Also, to measure B cells using flow cytometry, red blood cells need to be removed with lysis buffer, lysed cells removed by washing with centrifugation, and the lysis buffer neutralized. In the flow cytometry protocol, about 6 ml of phosphate buffered saline (PBS) is required to wash the sample, compared to 80 μl of PBS used in the gravity-based channel separation. As a result, good correlation data was shown to exist between the measurement of C4d deposition on B cells by conventional flow cytometry and the image analysis of C4d deposition on B cells separated using the gravity-based channel (Figure 29).

Example

[0189] Application of the microchannel cartridge of the present disclosure in the diagnosis of systemic lupus erythematosus ("SLE") Whole blood samples are collected from patients with or suspected of having systemic lupus erythematosus (SLE) and healthy control groups. The whole blood samples are labeled for gravity-based channel separation and detection of C4d on B cells as described in Example 3. The captured B cells are imaged and analyzed as described in Example 5, and the levels of C4d (such as cell-bound C4d) on B cells in samples from systemic lupus erythematosus (SLE) patients and healthy control groups are quantified.

[0190] Next, the C4d levels on B cells in samples from patients with systemic lupus erythematosus (SLE) are compared to those in a healthy control group to determine whether the patients' cell-bound C4d levels are elevated above the levels in the healthy control group (the "control levels"). The control levels may be the cell-bound C4d levels of individual healthy controls or the average cell-bound C4d level of the entire healthy control group. If the patients' cell-bound C4d levels are elevated and the difference between the patients' cell-bound C4d levels and the control levels is greater than a threshold level, the patient may be classified as having systemic lupus erythematosus (SLE). Such classification involves assigning a probability that the patient is likely to have systemic lupus erythematosus (SLE) and / or is likely to benefit from treatment for SLE.

Example

[0191] Comparison of a fluorescence-activated cell sorting system (FACS) and a cell separation device using gravity Table 4 shows a comparison between a fluorescence-activated cell sorting system (FACS) and an exemplary gravity-based cell separation device shown in FIGS. 7 to 21. First, in the exemplary gravity-based cell separation device, only 10 μl of whole blood is used, which is much less compared to the 50 μl required by the fluorescence-activated cell sorting system (FACS). Furthermore, the fluorescence-activated cell sorting system (FACS) requires a large amount of other reagents such as anti-CD20-FITC, anti-C4d Alexa 647, and red blood cell (RBC) lysis buffer. Currently, it takes 2 hours for the fluorescence-activated cell sorting system (FACS) to separate B cells and measure C4d levels, while it takes 2.5 hours for the gravity-based cell separation device. This 2.5-hour sample analysis time by the gravity-based cell separation device can be further shortened by optimizing the gravity separation time. Since the gravity-based cell separation device is a microchannel-based technology, the required amount of phosphate-buffered saline (PBS) washing buffer can be reduced to 1 / 100. Furthermore, there is a good correlation between the fluorescence-activated cell sorting system (FACS) and the gravity-based cell separation device in measuring the C4d level in patients with lupus, and the R 2 value shows 0.97.

[0192]

Table 4

Example

[0193] Cell separation by an exemplary gravity-based cell separation device equipped with a cubic magnet With the cell sorting device shown in FIGS. 26 to 33, the microchannel cartridge can be easily attached to and removed from the holding device without using rubber bands for sample loading, cell separation, and analysis. The dimensions of an exemplary cell separation device are 14 inches x 8 inches x 8 inches, and the weight is approximately 1 kg. The materials used in the manufacture of this cell separation device include wood, acrylate plastic, aluminum hinges, cube magnets, compression springs, and washers.

[0194] A small aliquot of 10 μl of whole blood was diluted 1:1 using a magnetic separation buffer (BD IMag® buffer from BD Biosciences) containing BSA and EDTA in phosphate-buffered saline (PBS). 1 μl of mouse anti-human CD19 magnetic particles, 3 μl of mouse anti-human CD20 FITC antibody (from BD Biosciences), and an anti-human C4d 9A10 biotin / streptavidin conjugate antibody (from BD Biosciences) with a nominal concentration (0.32 μg) were added to the diluted whole blood. The total volume of the whole blood and antibodies before magnetic separation was only 33 μl.

[0195] The fabricated microchannels were placed on a holding device, and 70 μl of PBS solution was injected. The injected PBS solution rapidly moved into the cartridge by capillary action. After adding the phosphate-buffered saline (PBS) solution, 30 μl of labeled blood sample solution was injected. After all the blood was loaded, the separation device was kept horizontal, and the blood sample was incubated in the dark for 25 minutes. This step is also called the first gravity separation. After the first gravity separation, the separation device was rotated by +90 degrees with respect to the horizontal line for gravity separation, and at this position, red blood cells (RBCs) moved downward toward the RBC collection chamber, while the target B cells were maintained in the upper magnet chamber. During this gravity separation, only biological particles such as cells and cell debris moved, and the fluid did not move. After gravity separation, the separation device was moved to the standby position at 0 degrees with respect to the horizontal line, and at this position, 80 μl of phosphate-buffered saline (PBS) was injected to wash away unbound fluorescent dyes and other unbound biological particles. After washing with phosphate-buffered saline (PBS), the cubic magnet was removed from the microchannel cartridge, and subsequently, the cartridge was removed from the holding device of the cell separation device using gravity for image analysis.

Example

[0196] Image analysis of cells using the magnets and magnet configurations of the present disclosure As will be further described below, it is necessary to reduce the number of images by condensing the cell capture region. This was achieved by changing the type and / or orientation of the magnet. When B cells are attracted to the condensed small region, a problem occurs in that the B cells overlap with other B cells and red blood cells (RBCs). The ImageJ plugin used to calculate the number of B cells and cell-bound C4d excludes overlapping cells. To test whether the image analysis algorithm can measure sufficient cells with only a few images and accurately measure cell-bound C4d, two samples with different levels of B cell-bound C4d were investigated using the exemplary magnets and magnet configurations shown in FIGS. 26 to 33. Images of the captured cells were taken and measured using the ImageJ plugin.

[0197] Seven images were taken of the cell capture region of sample 214632 and its surrounding area. The total number of cells obtained by image analysis in all seven images was 285 (Table 5). The algorithm calculated a C4d level of 113.4 for all 285 B cells and a C4d level of 113.2 for the 64 B cells collected in the above image. Five images of captured cells were taken from sample 214817, and the total number of B cells was 81. Since the total number of B cells was 81, the C4d level was 31.5, which was the same as the level calculated for 66 cells using one less image. From these results, it was confirmed that when the number of cells was about 50, a good correlation was obtained between image analysis and the fluorescence intensity of the fluorescence-activated cell sorting system (FACS), but this was achieved only by taking 25 to 30 images. There is an advantage that a sufficiently small area is generated by the new magnet and the orientation of the magnet, and the required number of images is reduced to 4 or 5.

[0198]

Table 5

Example

[0199] Further improvement of gravity separation (1) By pre-filling the microchannel of the cartridge with a phosphate-buffered saline (PBS) buffer, a longer separation path by gravity is created in the microchannel.

[0200] To improve the separation of blood samples in the microchannel, a method of increasing the effective separation path in the microchannel without physically increasing the dimensions of the cartridge (e.g., length, etc.) was tested. For example, in a microchannel pre-filled with a phosphate-buffered saline (PBS) buffer, to test whether it is possible to move red blood cells (RBCs) further downward by gravity, approximately 80% of the microchannel was pre-filled with phosphate-buffered saline (PBS), and the remaining 20% of the microchannel was occupied by a labeled blood sample.

[0201] When the phosphate buffered saline (PBS) buffer was not pre-filled, the separation trajectory due to gravity was only 20 mm. However, when the phosphate buffered saline (PBS) buffer was pre-filled, the separation trajectory was 70 mm, which was 3.5 times the length of the microchannel without pre-filling the phosphate buffered saline (PBS) buffer. By making the separation trajectory longer, it becomes possible to move red blood cells (RBCs) and other wastes to a location farther from the capture region of B cells.

[0202] (2) By using an improved type and configuration of magnet, B lymphocytes are concentrated in a smaller imaging field.

[0203] In the conventional system, a slab magnet (i.e., a magnet having a substantially flat rectangular shape) has been used to capture B cells. Due to the shape of the slab magnet, the captured B cells spread over a long magnetic surface. Because of the length of this magnetic surface, imaging takes a long time, and there may be only one or two B cells present in one image. To obtain a sufficient number of B cells for statistical analysis, it is necessary to image and identify B cells on hundreds of images, which is very burdensome in practice.

[0204] To solve this problem, a cube magnet was constructed to concentrate B cells in a small region within the microchannel. Instead of having its flat surface arranged parallel to the length of the microchannel, the cube magnet is oriented such that its vertices (i.e., its corners) face in the direction of the cell collection chamber. (See, for example, the orientation of cube 404 in Figure 21). With this configuration, an unexpectedly controllable imaging field (e.g., a circle of approximately 2 mm) could be realized. In such a smaller imaging field, only one or two images are needed to image and count all B cells. This can significantly save the time of imaging and checking a large number of images as has been done conventionally.

[0205] When using a flat magnet, in order to detect a sufficient number of B cells (50 - 100 B cells), it is necessary to search for B cells in a wide area of 10 mm x 12 mm from the microscope. In other words, the user has to measure or image B cells on approximately 174 computer screens of the microscope, and this operation takes at least 30 minutes. In contrast, when using a cube magnet, in order to obtain sufficient B cells, it is only necessary to search a circular area of 2 mm. The user only has to look at one or two computer screens of the microscope, thereby significantly saving the time for sample analysis. When using a cube magnet, the time required to detect all target B cells is less than 1 minute.

[0206] (3) Flow control during waste removal To capture more B cells, it is advantageous to use a low flow rate. B cells are ball-shaped and roll and move easily within microchannels. Therefore, even when using magnetic nanoparticles, the loss of B cells is more affected by the flow rate compared to RBCs (doughnut shape). In experiments, it was found that in dry filter paper, an initial burst occurred at a very high flow rate, resulting in a large loss of B cells. A lower flow velocity is better. To reduce the flow velocity, a method of evaporating phosphate-buffered saline (PBS) from a paper wick was evaluated. As shown in Fig. 25, phosphate-buffered saline (PBS) was wicked into the microchannel 2520 using a wet filter paper or a dry filter paper 2501. By drying the filter paper, the phosphate-buffered saline (PBS) was wicked at a low speed, and at the same time, tests were conducted for both room temperature and heated air to construct a waste removal system. Heated air 2503 was supplied with a heat gun to slowly dry the wick. As a result, it was found that more B cells could be retained by removing the phosphate-buffered saline (PBS) washing solution and waste at a low speed. In fact, better separation was possible and the collection amount of B cells increased by slowly removing the washing solution and waste with a wet filter paper and optionally room temperature air or heated air that slowly evaporates the liquid (such as water) from the wet filter paper.

[0207] In the above-mentioned evaporation-based flow rate control, a wet filter paper was used to avoid an increase in the initial burst flow rate. However, when using a wet filter paper, the microchannel becomes larger. To solve this problem, two types of wet filter papers were evaluated, such as a vertical wet filter paper and a flat filter paper horizontally arranged at the end of the microchannel. As a result, it was found that in any type of filter paper, it is effective in reducing the flow velocity and removing waste (such as excess fluorescent dye, unbound magnetic nanoparticles, red blood cells (RBC), etc.) from the microchannel. However, in terms of simplicity and waste removal ability, the flat filter paper is more excellent.

[0208] (4) Concentrate B cells on a magnet To achieve better separation, the magnetically labeled B cells underwent first and second gravity separation steps as shown in FIGS. 26A-26C. FIG. 26A shows the difference in the migration behavior of B cells in a microchannel when two different gravity separation methods were used. Path A shows cell migration when the first and second gravity separation steps were performed. During the first gravity separation period, the magnetically labeled B cells migrate towards the bottom slide of the horizontally placed microchannel. Next, when the microchannel is tilted 90 degrees vertically, the B cells deposited on the bottom slide of the microchannel begin to migrate towards the cube magnet. In this way, the B cells undergo a two-dimensional gravity separation process, that is, the first and second gravity separations that enable more B cells to be captured by the cube magnet. In contrast, Path B involves only the second gravity separation and no first gravity separation. Since there is no first gravity separation and only the second gravity separation, the B cells in the injected blood sample do not have enough time to reach the bottom slide of the microchannel. Therefore, many of the B cells passing through Path B do not have sufficient opportunity to approach the apex of the cube magnet. As a result, many of the B cells migrate to the exit of the microchannel, resulting in a large amount of cell loss. Considering that the size of B cells is 10 to 20 microns and the distance between the upper and bottom slide glasses of the microchannel used in this study is 160 microns, they are very small biological particles in the microchannel.

[0209] In this study, the conditions of the first gravity separation step were investigated. The microchannel was held horizontally for 0 to 35 minutes, and the cells were sedimented to the bottom of the microchannel while moving towards the magnet. Good separation was achieved after the first and second gravity separations, with the first gravity separation time being approximately 25 minutes and the second gravity separation time being approximately 54 minutes.

[0210] As shown in FIG. 26B, when the first gravity separation was not performed, only 93 B cells could be captured. However, the number of captured B cells increased as the first gravity separation time became longer until 25 minutes (287 captured B cells). After 35 minutes, the capture of B cells showed a stagnation, which means that 25 minutes is the optimal first separation time. As a conclusion, the B cells captured after 25 minutes in the first gravity separation step were three times as many as those when the first gravity separation step was not performed.

[0211] FIG. 26C shows the functional mode of two-dimensional gravity separation. This figure shows several steps including a blood sample injection step, a first horizontal gravity separation step, a second vertical gravity separation step, and a channel cleaning step. As shown in FIG. 26C, the microchannel slopes backward to wash the sample using a wet filter paper.

[0212] (5) Improved magnet and microchannel holding device The small cell concentration area above the cubic magnet may disperse when the microchannel is removed from the magnet. This frequently occurs when the microchannel is manually separated from the magnet. When this small area disperses, some of the captured B cells may escape from the small area, resulting in the loss of B cells. This problem also occurs with the rubber band used to fix the microchannel to the platform and the magnet. The microchannel becomes more likely to collide, and the microchannel may be moved on the magnet by the rubber band.

[0213] As shown in FIGS. 17 to 21, for an embodiment of the present method, an improved holding device having three independently moving panels was devised. This microchannel holding device maintained the condensed cell small region very well. The microchannel holding device of this embodiment uses three independently moving panels with hinges instead of a rubber band. As shown in FIG. 21, the bottom platform enables the magnet to be pulled away directly below the microchannel, thereby maintaining the cell capture small region. Also, in this design, an opening is provided below the microchannel to insert or move the imaging device to a predetermined position within a benchtop unit.

[0214] (6) Use of hot air to speed up the washing process After gravity separation, waste materials (i.e., excess fluorescent dyes, unbound magnetic nanoparticles, red blood cells (RBC), etc.) accumulate at the ends of the microchannels. These waste materials dry to form blood clots and block the exits of the microchannels, thus interfering with the washing process with phosphate-buffered saline (PBS) using wet filter paper.

[0215] To solve this problem, the exits of the microchannels were heated with hot air to break up the coagulated blood waste and move the waste towards the wet filter paper. Further, a razor blade was used to remove the dried blood at the exits of the microchannels. It was found that the hot air from the heat gun was very effective in breaking up and removing the dried coagulated blood waste. Also, the hot air was able to remove moisture from the wet filter paper, but this did not dramatically increase the flow rate. When hot air was used, the time required for the washing process was only 5 minutes. Further, by using room-temperature air in combination with the razor blade, the dried blood at the exits of the microchannels could be effectively broken up. With room-temperature air, the washing process took about 119 minutes.

[0216] (7) Use of a gravity-based cell separation device equipped with a microscope for mobile phones The cell separation device using gravity according to the present disclosure can be used in various configurations such as the following two configurations. (a) Remove the microchannel from the cell separation and holding device using gravity and move it to an arbitrary microscope inside. With this configuration, the use of the cell separation device can be expanded to both research and clinical applications. (b) Without removing the microchannel, push down the magnet holding device, turn the microchannel holding device upside down, slide the microscope attached to the mobile phone to the unit, and image it using a custom application (see FIG. 14). If necessary, the light source of the microscope can be incorporated into the unit.

[0217] The descriptions of the above-described embodiments and preferred embodiments are not intended to limit the invention defined in the claims, but should be construed as illustrative. As can be easily understood, numerous variations and combinations of the features described above can be utilized without departing from the invention defined in the claims. Such variations are not considered to be a departure from the scope of the invention, and all such variations are intended to be included in the following claims. All references cited herein are hereby incorporated by reference in their entirety.

Claims

1. A microchannel cartridge, comprising: a first layer; a second layer; a microfluidic channel layer having microchannels configured to separate cells or cell fragments in a fluid sample, the microfluidic channel layer being disposed between the first layer and the second layer, and the microchannels comprising: an inlet; an outlet; a first flow path in fluid communication with the inlet; a cell collection chamber provided downstream of the first flow path and in fluid communication with the first flow path; a second flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the outlet wherein the microfluidic channel layer has the following structure, The microchannel cartridge has: When the inlet is disposed below the outlet, the microchannel cartridge is configured such that the passage of the fluid sample into the second flow path is delayed, thereby enabling the fluid sample to be loaded into the inlet by gravity. When the inlet is disposed above the outlet, the microchannel cartridge is configured such that the passage of unnecessary cells or cell fragments into the second flow path is facilitated, thereby enabling the cells or cell fragments in the fluid sample to be separated by gravity. A microchannel cartridge.

2. The microchannel cartridge according to claim 1, further comprising a magnetic member disposed at or near the inlet, the magnetic member being configured to apply a magnetic force to the fluid sample received from the inlet.

3. The microchannel cartridge according to claim 1 or 2, wherein the magnetic member comprises a cubic magnet, and vertices of the cubic magnet are disposed below and facing the cell collection chamber.

4. The microchannel cartridge according to any one of claims 1 to 3, wherein the microfluidic channel layer has a polymer thin film comprising styrene-butadiene-styrene (SBS), styrene-isoprene-styrene (SIS), polypropylene coated with a pressure-sensitive adhesive, or a combination thereof.

5. The microchannel cartridge according to any one of claims 1 to 4, further comprising a pretreatment solution occupying at least 50% of the microchannel.

6. The microchannel cartridge according to any one of claims 1 to 5, wherein the pretreatment solution contains a phosphate buffered saline buffer.

7. The microchannel cartridge according to any one of claims 1 to 6, wherein the first layer or the second layer has a microscope slide glass.

8. The microchannel cartridge according to any one of claims 1 to 7, further comprising a waste chamber provided downstream of the cell collection chamber and in fluid communication with the second flow path.

9. The microchannel cartridge according to claim 8, further comprising a third flow path provided downstream of the cell collection chamber and in fluid communication with the cell collection chamber and the waste chamber.

10. The microchannel cartridge according to claim 9, wherein the width of the third flow path is larger than the width of the first flow path or the second flow path.

11. The microchannel cartridge according to any one of claims 1 to 10, wherein the second flow path has a meandering flow path or a straight flow path.

12. The microchannel cartridge according to any one of claims 1 to 11, further comprising a coating attached to at least a part of the first flow path and / or the second flow path.

13. In the microchannel cartridge according to claim 12, the coating includes chitosan (e.g., neutral chitosan, chitosan salt, chitosan derivative), chitin, polymethyl methacrylate (PMMA), silicone, polystyrene (PS), polysaccharide (e.g., nonionic, ionic, crosslinked polysaccharide), poly-D-lysine, streptavidin, collagen, polyurethane, epoxy, or a combination thereof. The microchannel cartridge is as described above.

14. In the microchannel cartridge according to any one of claims 1 to 13, the coating includes one or more antibodies. The microchannel cartridge is as described above.

15. In the microchannel cartridge according to claim 14, the one or more antibodies are associated with quantum dots or are biotinylated. The microchannel cartridge is as described above.

16. In the microchannel cartridge according to claim 14, the one or more antibodies include an anti-C4d antibody or an antibody that specifically binds to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268. The microchannel cartridge is as described above.

17. In the microchannel cartridge according to any one of claims 1 to 16, the first flow path or the second flow path has a height of about 50 μm to about 500 μm. The microchannel cartridge is as described above.

18. In the microchannel cartridge according to any one of claims 1 to 17, the first flow path or the second flow path has a width of about 2 mm to about 20 mm. The microchannel cartridge is as described above.

19. In the microchannel cartridge according to any one of claims 1 to 18, the microchannel has a length of about 25 mm to about 75 mm. The microchannel cartridge is as described above.

20. In the microchannel cartridge according to any one of claims 1 to 19, the cell collection chamber has a rectangular, elliptical, or rhombic shape, the microchannel cartridge.

21. In the microchannel cartridge according to claim 20, the cell collection chamber has a rhombic shape, the microchannel cartridge.

22. In the microchannel cartridge according to claim 20 or 21, the cell collection chamber is about 9 mm ２ to about 225 mm ２ and has an area of, the microchannel cartridge.

23. In the microchannel cartridge according to any one of claims 1 to 22, the inlet or the outlet has an absorbent material disposed therein, the microchannel cartridge.

24. In the microchannel cartridge according to claim 23, the absorbent material has a pore diameter in the range of about 100 μm to about 500 μm, the microchannel cartridge.

25. In the microchannel cartridge according to claim 23 or 24, the absorbent material has absorbent fibers or a sponge, the microchannel cartridge.

26. In the microchannel cartridge according to any one of claims 23 to 25, the absorbent material includes cotton, polyester, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), or a combination thereof, the microchannel cartridge.

27. In the microchannel cartridge according to any one of claims 23 to 26, the absorbent material is configured to generate a capillary force greater than that of the second flow path to prevent air bubbles from entering the inlet, the microchannel cartridge.

28. In the microchannel cartridge according to any one of claims 23 to 27, the microchannel cartridge has a wet filter paper in fluid communication with the outlet, the microchannel cartridge.

29. In the microchannel cartridge according to any one of claims 1 to 28, the fluid sample includes whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, plasma, or a combination thereof, the microchannel cartridge.

30. In the microchannel cartridge according to any one of claims 1 to 29, the fluid sample contains blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets. A microchannel cartridge.

31. In the microchannel cartridge according to any one of claims 1 to 30, the fluid sample contains magnetically labeled cells. A microchannel cartridge.

32. A kit comprising: One or more microchannel cartridges according to any one of claims 1 to 31, Optionally a buffer or instruction materials And a kit.

33. The kit according to claim 32, further comprising an immunological reagent. A kit.

34. The kit according to claim 33, wherein the immunological reagent contains an antibody. A kit.

35. A microchannel cell separator comprising: One or more microchannel cartridges according to any one of claims 1 to 31, A rotating member configured to change the angle of the microchannel cartridge, whereby the flow rate of the fluid sample in the microchannel is adjusted. The rotating member And a Microchannel cell separator.

36. In the microchannel cell separator according to claim 35, the rotating member has a holding device for the microchannel cartridge, and the microchannel cartridge is detachably attached to the holding device. A microchannel cell separator.

37. In the microchannel cell separator according to claim 35 or 36, the rotating member has an angle indicator having one or more marks indicating a loading position, a standby position, and / or a sorting position. A microchannel cell separator.

38. In the microchannel cell separator according to any one of claims 35 to 37, the rotating member is configured to rotate the microchannel cartridge continuously or intermittently in order to control the flow rate of the fluid sample. A microchannel cell separator.

39. In the microchannel cell separation device according to any one of claims 35 to 38, the rotating member is driven by a motor, the microchannel cell separation device.

40. In the microchannel cell separation device according to any one of claims 35 to 39, further comprising a base for supporting the rotating member, the microchannel cell separation device.

41. A method for separating cells or cell fragments in a fluid sample, The step of arranging the microchannel cartridge according to any one of claims 1 to 31 at a sample loading angle; The step of introducing the fluid sample into the microchannel through the inlet of the microchannel; The step of performing a first gravitational separation by incubating the fluid sample for a first period; The step of arranging the microchannel cartridge at a sorting angle for gravitational separation for a second period to perform a second gravitational separation, wherein the flow of the target cells or cell fragments is induced into the cell collection chamber by this step, the step of performing the second gravitational separation A method having.

42. In the method according to claim 41, further Before the step of introducing the fluid sample, a step of loading a pretreatment solution into the microchannel, a method.

43. In the method according to claim 42, the pretreatment solution is selected from distilled water, deionized water, and phosphate buffered saline buffer, the method.

44. In the method according to claim 43, the cleaning solution contains a phosphate buffered saline buffer, the method.

45. In the method according to claim 41 or 44, further After the step of introducing the fluid sample, a step of loading a cleaning solution into the microchannel to clean the cells or the cell fragments, a method.

46. In the method according to claim 45, further After the step of washing the cells or the cell fragments, a step of arranging a wet absorbent material at the outlet, the wet absorbent material being in fluid communication with the outlet and facilitating the removal of unnecessary cells or cell fragments, a method.

47. In the method according to claim 46, the wet absorbent material is wet filter paper, the method.

48. In the method according to claim 46, further A method having a step of applying hot air or room temperature air to the wet absorbent material to gradually evaporate liquid from the wet absorbent material in order to facilitate removal of unnecessary cells or cell fragments.

49. In the method according to any one of claims 41 to 49, further comprising A method having a step of adding an immunological reagent to the fluid sample before the step of introducing the fluid sample.

50. In the method according to claim 49, the immunological reagent contains one or more antibodies.

51. In the method according to claim 50, the one or more antibodies are associated with quantum dots or are biotinylated.

52. In the method according to claim 50 or 51, the one or more antibodies contain an anti-C4d antibody or an antibody that specifically binds to CD3, CD4, CD5, CD8, CD45, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD42b, CD69, CD70, CD79, CD80, CD85, CD86, CD137, CD138, CD252, or CD268.

53. In the method according to any one of claims 41 to 52, the fluid sample contains whole blood, washed red blood cells or cell fragments thereof, concentrated red blood cells or cell fragments thereof, platelets or cell fragments thereof, serum, or plasma.

54. In the method according to any one of claims 41 to 53, the fluid sample contains blood cells selected from red blood cells, reticulocytes, T lymphocytes, B lymphocytes, monocytes, granulocytes, eosinophils, basophils, and platelets.

55. In the method according to any one of claims 41 to 54, the fluid sample contains magnetically labeled cells.

56. In the method according to any one of claims 41 to 55, the sample loading angle is an angle of about -90 degrees to about 0 degrees below the horizontal line.

57. In the method according to any one of claims 41 to 56, the sample loading angle is an angle of about 90 degrees to about 0 degrees above the horizontal line.

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